Protein complexes having factor VIII:C activity and production thereof

ABSTRACT

Recombinant protein complexes having human Factor VIII:C activity are expressed in a eukaryotic host cell by transforming the host cell with first and second expression cassettes encoding a first polypeptide substantially homologous to human Factor VIII:C A domain and a second polypeptide substantially homologous to human Factor VIII:C C domain, respectively. In the present invention, the first polypeptide may be extended having at its C-terminal a human Factor VIII:C B domain N-terminal peptide, a polypeptide spacer of 3-40 amino acids, and a human Factor VIII:C B domain C-terminal peptide. Expression of the second polypeptide is improved by employing an  alpha 1-antitrypsin signal sequence.

CROSS-REFERENCE TO RELATED APPLICATION

This application is a continuation of application Ser. No. 07/652,099filed on 7 Feb. 1991, which is a continuation-in-part of applicationSer. No. 07/051,915 filed 19 May 1987, which is a continuation-in-partof application Ser. No. 06/822,989 filed 27 Jan. 1986, now abandoned.

DESCRIPTION Technical Field

This invention relates to protein complexes having Factor VIII:Cactivity, and to methods for producing said complexes by expression ofsuitable polynucleotide constructs. The protein complexes are useful inthe treatment of classical (Type A) hemophilia.

BACKGROUND OF THE INVENTION

Hemophilia A is an X-chromosome-linked inherited disease which afflicts1-2 males per 10,000. The disease is caused by an absence or deficiencyof Factor VIII:C. Factor VIII:C is a very large glycoprotein (nativeM_(r) 330 K-360 K), which is present in plasma at extremely lowconcentrations. It is a necessary element in the proteolytic cascadewhich converts soluble fibrinogen to insoluble fibrin, forming a clot toprevent blood loss from traumatized tissue. In the bloodstream, it isfound in noncovalent association with Factor VIII:R ("von Willebrandfactor"), which acts as a stabilizing carrier protein. Factor VIII:C isvery susceptible to cleavage by thrombin, plasmin, protease C, and otherserine proteases. It is generally isolated from plasma or plasmaproducts as a series of related polypeptides ranging from M_(r) 160 K-40K with predominant species of M_(r) 92 K and M_(r) 80 K-77 K. Thiscomplex pattern has made the analysis of the structure of active FactorVIII:C very difficult.

Factor VIII:C and the related polypeptides have been described by F.Rotblat et al, Biochemistry (1985)24:4294-4300; G. A. Vehar et al,Nature (1984) 312:337-342; J. J. Toole et al, Nature (1984) 312:342-347;and M. A. Truett et al, DNA (1985) 4:333-349. E. Orr et al, MolecularGenetics of Clotting Factors, p. 54, s321, reported a "spacer" functionfor the heavily glycosylated region of Factor VIII:C. The sequence hasbeen reported by J. J. Toole et al, supra; W. I. Wood et al, Nature(1984) 312:330-336; and M. A. Truett et al, supra. The full-lengthprotein contains three repeats of one sequence (I), and two repeats of asecond sequence (III). A third, heavily glycosylated sequence (II) ispresent between the second and third I repeats, and is apparentlycleaved proteolytically to form the M_(r) 92 K and M_(r) 80 Kpolypeptides. The first two I repeats form the A domain, while the thirdI repeat and the two III repeats form the C domain. The II sequenceforms the B domain. Thus, the full-length protein has the structure I₂-I₂ -II-I₃ -III₁ -III₂ (A-B-C), while the M_(r) 92 K and M_(r) 80 Kpolypeptides (A and C) have the structures I₁ -I₂ and I₃ -III₁ -III₂,respectively. C. Fulcher et al, J Clin Invest (1985) 76:117-124,suggested that based on antibody-epitope data with Factor VIII:C, boththe M_(r) 92 K and the M_(r) 80 K polypeptides are necessary for FactorVIII:C function.

Factor VIII:C has historically been isolated from blood in aconcentrated form for therapeutic treatment of hemophilia. However,concerns regarding transmission of HIV and other blood-borne diseaseshave stimulated activity to provide alternative supplies of FactorVIII:C. It is of substantial interest to be able to supply compositionshaving Factor VIII:C activity without concerns as to the transmissionviral diseases associated with the native Factor VIII:C.

Although full-length recombinant human Factor VIII:C has been produced,it is difficult to purify and characterize, and it is unstable due toproteolysis. Efficient recombinant production of the full-lengthmolecule for clinical use is doubtful at this time.

R. L. Burke et al, J Biol Chem (1986) 261:12574-78 disclosed theexpression of an active Factor VIII:C complex from cells simultaneouslytransfected with polynucleotides encoding M_(r) 92 K and M_(r) 80 Kpolypeptides. The obtained protein demonstrated activity equal to thatof cloned full-length Factor VIII:C expressed under similar conditions.O. Nordfang et al, J Biol Chem (1988) 263:1115-18 disclosed the in vitroassembly of active Factor VIII:C complexes from separate preparations ofM_(r) 92 K protein and M_(r) 80 K protein (FVIII-HC and -LC,respectively). Successful assembly required divalent metal ions(especially Mn⁺⁺ and Ca⁺⁺ ) and thiols, but only a small amount ofFVIII-HC could be complexed into active FVIII:C.

DISCLOSURE OF THE INVENTION

We have now invented an improved method for expressing recombinantprotein complexes with high stability and Factor VIII:C activity. TheM_(r) 92 K polypeptide (FVIII-HC) and the M_(r) 80 K polypeptide(FVIII-LC) are expressed as two separate polypeptides, under the controlof separate promoters, within the same host cell. Each polypeptide ispreferably expressed using a signal sequence which directs export to theextracellular space with cleavage of the signal sequence. FVIII-HC ispreferably expressed as a fusion protein having a C-terminal extension.The extension comprises a polypeptide sequence homologous to the Bdomain N-terminal sequence (which may allow cleavage by thrombin), apolypeptide spacer of 3 to 100 amino acids, and a sequence homologous tothe C-terminal B domain sequence. The C-terminal extension of FVIII-HCresults in a higher yield of active polypeptide upon expression ineukaryotic host cells. FVIII-LC is preferably expressed as an LCpolypeptide using a signal peptide. The FVIII-LC polypeptide isprocessed and secreted efficiently with the correct N-terminal aminoacid residue, and correct glycosylation. Co-transfection withpolynucleotides encoding FVIII-HC and FVIII-LC in a suitable host cellprovides recombinant protein complexes having Factor VIII:C activity inhigh yield.

MODES OF CARRYING OUT THE INVENTION

A. Definitions The term "polynucleotide" as used herein refers to asequence of DNA or RNA, which may be single or double-stranded (ss ords), or a DNA-RNA heteroduplex. In most cases herein, polynucleotidewill refer to dsDNA.

The term "signal peptide" as used herein refers to a peptide sequencewhich is recognized and acted upon by signal peptidase during expressionof the polypeptide. Signal peptides encode peptide sites for signalpeptidase cleavage, and cause the attached polypeptide to be transportedinto the secretion pathway leading to the extracellular medium.

The term "A domain" refers to that portion of human Factor VIII:C whichconstitutes the M_(r) 92 K protein subunit. The A domain contains fromabout 740 to about 760 amino acids, and is found at the N-terminus ofthe native human Factor VIII:C. The A domain polypeptide will extendfrom amino acid 10, usually amino acid 1, to at least about amino acid620, usually at least about amino acid 675, more usually at least aboutamino acid 740. The polypeptide will include at least about 85% of the Adomain (Wood et al, supra), more usually at least about 90% and mayoptionally include a portion of the N-terminus of the B domain,typically not exceeding about amino acid 1405. Of particular interest isan N-terminal chain having the entire sequence to the thrombolyticcleavage site at Arg₇₄₀ -Ser₇₄₁.

The term "B domain" refers to that portion of native human Factor VIII:Cwhich is generally removed by intracellular cleavage, and which isheavily glycosylated when expressed in mammalian cells such as COS7 andCHO. The B domain contains an N-terminal sequence, which allows cleavageof the A domain from the B domain by thrombin. The B domain also has aC-terminal processing site which allows cleavage of the C domain fromthe A-B precursor by an enzyme located in the Golgi apparatus of themammalian cell. The sequences of the N-terminal and C-terminal sequencesare set forth in the Examples below. The complexes of the inventionwhich lack "a substantial portion of the B domain" lack all of the Bdomain, or essentially all of the B domain except for the N-terminal andC-terminal sequences.

The term "C domain" refers to that portion of native human Factor VIII:Cwhich constitutes the C-terminus of the full length protein, and iscleaved intracellularly to form the Factor VIII:C light chain. The lightchain will have an amino acid sequence substantially the same as theamino acid sequence of the C-terminus of a Factor VIII:C polypeptide,usually at least about 80%, more usually at least about 90% of theFactor VIII:C M_(r) 80 K chain, particularly beginning with amino acid1570, usually amino acid 1600, particularly amino acid 1625, moreparticularly amino acid 1640, preferably at about amino acid 1649, ±10amino acids, more particularly ±1 amino acid, and continuing to at leastabout amino acid 2300, usually 2310, ±10 amino acids, preferably 2325,±5 amino acids, more preferably to the terminal amino acid (2332).Usually, the light chain will have at least about 85%, more usually atleast 95%, of the C₁ -C₂ domains, desirably the A₃ -C1-C2 domains.

The term "co-expressing" as used herein refers to simultaneousexpression of an A domain polypeptide and a C domain polypeptide withinthe same host cell. The polynucleotide sequences encoding the A and Cdomains may be on the same or on different expression cassettes orplasmids. Co-expression of the A and C domains permits proper folding tooccur, which in turn provides an A-C complex having higher activity andefficiency of secretion.

The term "cell growth medium" as used herein refers to any mediumsuitable for culturing host cells, and includes media suitable forobtaining expression of recombinant products whether actual cell"growth" occurs or not. Cell growth media generally include nutrientsand a metabolizable energy source in an aqueous solution. If desired,cell growth media may also include a compound which induces expressionof the recombinant polypeptides of the invention. Selection of such aninducing compound depends upon the promoter selected to controlexpression. Other typical additives include selection compounds (i.e.,drugs or other chemicals added to the media to insure that onlytransformed host cells survive in the medium) and serum, such as fetalbovine serum (FBS). "Serum-free medium" is a solution which has beensupplemented to such an extent that the necessary trace factors presentin serum need not be added in the form of serum. There are many suitablecell growth media available from commercial sources.

The term "polypeptide spacer" refers to a polypeptide sequence of about3 to about 100 amino acids, which is generally not homologous to thehuman Factor VIII:C B domain, and which carries fewer than 5 potentialsites of N-linked glycosylation. Preferably, there will be 2 or fewersuch sites. It is presently believed that the large size and high degreeof glycosylation of the B domain prevents efficient expression of theM_(r) 92 K polypeptide. However, a low (but useful) yield of the M_(r)92 K polypeptide is obtained when the B domain is completely removed. Itis also presently believed that the A domain may not be folded correctlyon a consistent basis in the absence of the B domain, so that only asmall percentage of the A domain is correctly folded and expressed. Thepolypeptide spacer of the invention provides a C-terminal extension tothe A domain, and apparently stabilizes the polypeptide and improvessecretion in active form. Thus, it may be that use of a polypeptidewhich is glycosylated lightly (or not at all) prevents the Adomain-spacer construct from encountering the same size problemsobstructing expression of full-length Factor VIII:C. The presentlypreferred spacer is derived from a human Ig heavy chain hinge,particularly from human IgA1. This spacer provides a flexible extension,without adding an immunogenic epitope (when administered in humans).

The term "homology" as used herein means identity or substantialsimilarity between two polynucleotides or two polypeptides. Homology isdetermined on the basis of the nucleotide or amino acid sequence of thepolynucleotide or polypeptide. In general terms, usually not more than10, more usually not more than 5 number %, preferably not more thanabout 1 number % of the amino acids in the chains will differ from theamino acids naturally present in the Factor VIII:C A and C domains.Particularly, not more than about 5%, more usually not more than about1% will be nonconservative substitutions. Conservative substitutionsinclude:

    ______________________________________                                        Gly ←→ Ala;                                                                          Val ←→ Ile ←→Leu;                  Asp ←→ Glu;                                                                          Lys ←→ Arg;                                    Asn ←→ Gln; and                                                                      Phe ←→ Trp ←→ Tyr.                 ______________________________________                                    

Nonconservative changes are generally substitutions of one of the aboveamino acids with an amino acid from a different group (e.g.,substituting Asn for Glu), or substituting Cys, Met, His, or Pro for anyof the above amino acids.

The term "sufficient amount" of protein complex of the invention refersto that amount of protein which is capable of effecting therapeutictreatment of a subject having a disorder treatable with native humanFactor VIII:C. In general, the protein complex of the invention isessentially as active as native human Factor VIII:C, and may beadministered in similar amounts. The specific activity of the proteincomplex of the invention may be determined by means known in the art, asdescribed below (e.g., by using the commercially available COATESTassay).

The term "effective concentration" refers to a concentration ofexpression cassette which is capable of transforming a host cell underappropriate transformation conditions.

B. General Method

DNA constructs are generally employed for expression of the polypeptidesof the invention. Each of the polynucleotide constructs will have, inthe 5'-3'-direction of transcription, a transcriptional initiation andtranslational initiation region, a structural gene coding regioncomprising a sequence coding for the signal peptide sequence, and asequence coding for the Factor VIII:C heavy or light chains, followed bytranslational and transcriptional termination sequences. The selectionof specific elements such as these is within the skill of the art.

The initiation region may comprise a number of different sequencesrelated to the initiation of transcription and translation. Thesesequences include enhancer sequences, RNA polymerase binding site, RNAcapping site, ribosomal binding and translational initiation sites, andthe like. The transcriptional initiation region may be the naturalregion associated with Factor VIII:C, or may be an alternative sequenceto provide for higher transcriptional efficiency. The sequences may beobtained from mammalian viruses or the genes of the host cell or genesfrom a different mammalian host which are active in the host cell.Numerous transcriptional initiation regions have been isolated anddemonstrated to be operative in mammalian host cells. These regionsinclude the SV40 early promoter and late promoter regions, theadenovirus major late promoter region, actin promoter region, thecytomegalovirus M_(r) 72 K immediate early protein promoter region, themetallothionein promoter, and the like.

The termination region may include 3'-untranslated sequences, apolyadenylation signal sequence, and the like. The termination regionmay be obtained from the 3' non-translated sequence of the Factor VIII:Cnatural cDNA, or may be from the same structural gene or differentstructural gene from which the 5'-initiation region was obtained. The3'-region is not as essential to the level of transcription as theinitiation region, so that its choice is more of a matter of conveniencethan specific selection.

The structural genes typically include a leader sequence coding for thesignal peptide which directs the polypeptide into the lumen of theendoplasmic reticulum for processing and maturation. Optionally includedare additional sequences encoding propeptides which are processedpost-translationally by endopeptidases, where the endopeptidases cleavea peptide bond, removing the propeptide to generate the maturepolypeptide. The signal peptide may be the naturally occurring one,particularly for the N-terminal peptide, or may be any signal peptidewhich provides for the processing and maturation of the polypeptides.

Various signal peptides have been reported in the literature and includesuch sequences as that of tissue plasminogen activator, immunoglobulinheavy and light chains, viral membrane glycoproteins such as HerpesSimplex virus glycoproteins gB and gD, α₁ -antitrypsin, and the like.The α₁ -antitrypsin signal peptide is presently preferred for secretionof the FVIII-LC polypeptide.

The DNA sequences encoding the mature protein and signal peptide must bejoined so as to be in reading frame. Where convenient restriction sitesare available, the cohesive or blunt ends may be properly joined.However, for the most part, adapters will be employed where portions ofthe coding sequence will be recreated in the synthetic adaptor so thatthe truncated structural gene and/or truncated signal sequence will belinked through the adaptor, so as to be in proper reading frame. Thesignal sequence and structural gene may be partially restriction mapped,so as to identify restriction sites, particularly unique restrictionsites, which may be employed to link the two sequences together inproper reading frame by means of an appropriate adaptor. Alternativelyunique restriction sites may be inserted at the junction of the signalsequence and mature polypeptide coding sequence by in vitro mutagenesis.

The translational start and stop signals will normally be part of thestructural gene, providing for the initiation codon at the beginning oftranslation and one or more stop codons for the termination oftranslation. The initiation codons will be the first codons of thesignal sequences. The stop codons may be added as appropriate as part ofthe termination region or be added to the coding region to provide forconvenient 3'-terminus for linkage to the transcriptional terminationregion to provide for a complete termination region.

The various regions of the expression cassette, (the transcriptional andtranslational initiation region nucleic acid sequence, structural genenucleic acid sequence encoding one of the polypeptides and under thetranscriptional and translational control of the initiation region, anda transcriptional and translational termination region, controlling theprocessing of the mRNA and the translational termination) which identifythe particular nucleotide sequences may be joined using conventionalmethods. Usually, the sequences obtained will contain, or be modified tocontain restriction sites, which may then be annealed wherecomplementary overhangs or cohesive ends are present. Modificationfrequently will be in noncoding regions by the introduction of linkersto provide for the desired cohesive ends. The ends will usually beligated prior to introduction into the host cell, although the host cellmay be allowed to provide the necessary ligation.

The expression cassettes may be joined to a wide variety of othersequences for particular purposes. Where amplification of the amount ofsecreted glycoprotein is desired, the expression cassettes for FVIII:Cmay be joined in tandem to a gene for which spontaneous increases ingene copy number can be selected by an appropriate treatment. Such genesinclude the human metallothionein gene, and the mouse dihydrofolatereductase gene. These genes are placed in cassettes having their owntranscriptional and translational regulatory sequences. By selectingcell clones resistant to increasing concentrations of heavy metal ions(e.g., cadmium) or methotrexate, the gene of interest (the expressioncassette) may be co-amplified in the host cell.

The subject expression cassettes may be part of a vector comprising areplication system functional in the host cell, which replication systemmay provide for stable episomal maintenance or integration of theexpression cassette into the host genome. The vector will also comprisea marker for selection, for selecting mammalian host cells containingthe DNA construct and the vector from those host cells which lack theDNA construct and vector.

A wide variety of replication systems are available, typically derivedfrom viruses that infect mammalian host cells. Illustrative replicationsystems include the replication systems from Simian virus 40,adenovirus, bovine papilloma virus, polyoma virus, Epstein Barr virus,and the like.

Selection markers enabling propagation of the vector in prokaryotic hostcells may include resistance to a biocide, particularly an antibiotic,or complementation of auxotrophy to provide a prototrophic host.Particular genes of interest as markers include kanamycin resistancegene (NPTII), chloramphenicol resistance gene (CAT), penicillinase(β-lactamase), or the like.

The vector will usually be circular, and will have one or morerestriction sites which allow for the insertion of the expressioncassette, stepwise or as a completed entity, into the vector.Frequently, the vector will also include a bacterial replication andselection system, which allows for cloning after each of themanipulative steps. In this way, relatively large amounts of theconstruction at each of the stages may be prepared, isolated, purified,tested to verify that the proper joining has occurred, and then used forthe next stage.

Various mammalian host cells may be employed in which the regulatorysequences and replication system are functional. Such cells include COS7cells, Chinese hamster ovary (CHO) cells, mouse kidney cells, hamsterkidney cells, HeLa cells, HepG2 cells, or the like.

The expression cassettes of the desired polypeptides may be linkedtogether in one nucleic acid chain or may be provided in separatenucleic acid molecules. The expression cassettes may be parts ofdifferent vectors or of the same vector. This is primarily a matter ofconvenience, although in some situations with particular vectors, one orthe other manner of construction may be preferable.

The expression vector may be a replication-deficient retrovirus. S. -F.Yu et al, Proc Nat Acad Sci USA (1986) 83: 3194-98 disclosed theconstruction of self-inactivating ("SIN") retroviral gene transfervectors. SIN vectors are created by deleting the promoter and enhancersequences from the U3 region of the 3' LTR. A functional U3 region inthe 5' LTR permits expression of the recombinant viral genome inappropriate packaging cell lines. However, upon expression of itsgenomic RNA and reverse transcription into cDNA, the U3 region of the 5'LTR of the original provirus is deleted, and is replaced with the U3region of the 3' LTR. Thus, when the SIN vector integrates, thenon-functional 3' LTR U3 region replaces the functional 5' LTR U3region, and renders the virus incapable of expressing the full-lengthgenomic transcript.

The expression cassettes are introduced into the host cell byconventional methods. Conveniently, calcium phosphate-precipitated DNAor DNA in the presence of DEAE-dextran may be employed fortransformation. A synthetic lipid particularly useful for polynucleotidetransfection is N- 1-(2,3-dioleyl-oxy)propyl!-N,N,N-trimethylammoniumchloride, which is commercially available under the name Lipofectin®(available from BRL, Gaithersburg, Md.), and is described by P. L.Felgner et al, Proc Nat Acad Sci USA (1987) 84:7413. Where viruses areinvolved, transfection or transduction may be employed. The particularmanner in which the host cell is transformed is not critical to thisinvention, depending substantially upon whether the expression cassettesare joined to a replication system and the nature of the replicationsystem and associated genes.

The transformed/transfected cells are then grown in an appropriatenutrient medium. It is presently preferred to employ CHO cells culturedat 10°to 32° C., more preferably about 27°C., for less than about 30hours, more preferably less than 10, most preferably less than about 4hours. The product is obtained as a complex of the two FVIII:C chains,so that the media or cell lysate may be isolated and the Factor VIII:Cactive complex extracted and purified. Various means are available forextraction and purification, such as affinity chromatography, ionexchange chromatography, hydrophobic chromatography, electrophoresis,solvent-solvent extraction, selective precipitation, and the like. Theparticular manner in which the product is isolated is not critical tothis invention, and is selected to minimize denaturation or inactivationand maximize the isolation of a high-purity active product.

Compositions are provided where the composition in the COATEST assaywill have at least 0.02 U/mL of activity, usually at least about 0.2,more usually at least about 0.5 U/mL of activity. The subject productcan be purified by affinity chromatography using antibodies,particularly monoclonal antibodies directed against the FVIII-LC,electrophoresis, extraction, HPLC, etc.

The subject method provides for production of a complex of the heavy andlight chains which has Factor VIII:C activity. Production is evidencedby conditioned media as described in the experimental section, whichwill have at least about 50, usually at least about 70 mU/mL, moreusually at least about 200 mU/mL of Factor VIII:C activity in theCOATEST assay.

The complexes having Factor VIII:C activity produced according to theinvention have a variety of uses as immunogens for the production ofantibodies, for isolation of von Willebrand factor by affinitychromatography, in diagnostic assays for Factor VIII:C and for treatmentof hemophiliacs and other hosts having blood clotting disorders. Thesubject protein complexes may be administered in a physiologicallyacceptable carrier, such as water, saline, phosphate buffered saline,and citrate buffered saline, at concentrations in the range of about10-200 U/mL. See U.S. Pat. Nos. 3,631,018; 3,652,530, and 4,069,216 formethods of administration and amounts. Other conventional additives mayalso be included.

C. Examples

The examples presented below are provided as a further guide to thepractitioner of ordinary skill in the art, and are not to be construedas limiting the invention in any way.

Example 1 (Preparation of Expression Plasmids)

(A) pSV7d:

The expression cassettes were prepared using the mammalian cellexpression vector pSV7d (2423 bp).

The plasmid pSV7d (see Truett et al, supra) was constructed as follows:The 400 bp BamHI/HindIII fragment containing the SV40 origin ofreplication and early promoter was excised from pSVgtI (obtained fromPaul Berg, Stanford University, California) and purified. The 240 bpSV40 BclI/BamHI fragment containing the SV40 polyA addition site wasexcised from pSV2/DHFR (Subramani et al, Mol Cell Biol (1981) 1:854-864)and purified. The fragments were fused through the following linker:##STR1## This linker contains five restriction sites, as well as stopcodons in all three reading frames. The resulting 670 bp fragmentcontaining the SV40 origin of replication, the SV40 early promoter, thepolylinker with stop codons and the SV40 poly-adenylation site wascloned into the BamHI site of pML, a pBR322 derivative having about 1.5Kb deleted (Lusky and Botchan, Cell (1984) 36:391), to yield pSV6. TheEcoRI and EcoRV sites in the pML sequences of pSV6 were eliminated bydigestion with EcoRI and EcoRV, treated with Bal31 nuclease to removeabout 200 bp on each end, and finally religated to yield pSV7a. TheBal31 resection also eliminated one BamHI restriction site flanking theSV40 region, approximately 200 bp away from the EcoRV site. To eliminatethe second BamHI site flanking the SV40 region, pSV7a was digested withNruI, which cuts in the pML sequence upstream from the origin ofreplication. This was recircularized by blunt end ligation to yieldpSV7b.

pSV7c and pSV7d represent successive polylinker replacements. First,pSV7b was digested with StuI and XbaI. Then, the following linker wasligated into the vector to yield pSV7c: ##STR2## Thereafter, pSV7c wasdigested with BglII and XbaI, and then ligated with the following linkerto yield pSV7d: ##STR3## (B) pSVF8-92:

pSVF8-92 is an expression plasmid for the M_(r) 92 K FVIII-HC chain.Starting from the BamHI site in the polylinker pSV7d, pSVF8-92 consistsof a 49 bp synthetic linker-adaptor molecule from BamHI to SacI encodingnucleotides -30 to +14 of the Factor VIII:C CDNA, (numbering from thefirst A of the translational start site; the sequence is shown below in(D) a 2267 bp SacI to HindIII fragment from the Factor VIII:C DNAcontained in pSVF8-200 described below (up to nucleotide +2281), andpSV7d from HindIII to BamHI.

(C) pSVF8-80:

pSVF8-80 is an expression plasmid for the M_(r) 80 K FVIII-LC chain.Starting from the SalI site in the polylinker pSV7d, pSVF8-80 consistsof a 201 bp fragment of a tissue plasminogen activator cDNA fromnucleotides -98 to +103 (relative to the start codon) terminating at aBglII site (tPA sequences given in S. J. F. Degan et al, J Biol Chem(1986) 261:6972-6985), a 29 bp synthetic BglII to BclI linker-adaptorencoding nucleotides +5002 to +5031 of Factor VIII:C ligated to a 2464bp BclI fragment of factor VIII:C spanning from a BclI site created atnucleotide 5028 of the factor VIII:C cDNA through in vitro mutagenesis(Zoller and Smith, Meth Enzymol (1983) 100:468) (pF8GM7), to a BclI sitein the 3' untranslated region, at nucleotide 7492, and a 400 bp fragmentof tPA 3' untranslated sequence spanning from a BglII site to asynthetic PstI site generated from the cDNA cloning, followed by thepolylinker from the vector M13mp9 (Vieira and Messing, Gene (1982)19:259) and then pSV7d.

(D) pSVF8-200

The vector pSVF8-200 is an expression plasmid for the full-length FactorVIII:C cDNA. The plasmid pSVF8-200 (described in Truett et al), whichcontains the entire Factor VIII:C CDNA coding and 3' untranslatedsequences, with the 5' untranslated sequences the same as describedabove for pSVF8-92, was prepared as follows.

Plasmid pSV7d was digested with BamHI to cut in the polylinker regiondownstream of the SV40 early promoter. The following 49 bp BamHI-SacIlinker adaptor, which codes for the last 30 bp of the 5' untranslatedregion and the first 15 bp of the human Factor VIII:C coding sequence,was chemically synthesized and ligated to pSV7d.

    __________________________________________________________________________      -35  -30  -25  -20  -15  -10  -5                                            5'                                                                               GATCC                                                                             TCTCC                                                                              AGTTG                                                                              AACAT                                                                              TTGTA                                                                              GCAAT                                                                              AAGTC                                         3'                                                                              BamHI G                                                                            AGAGG                                                                              TCAAC                                                                              TTGTA                                                                              AACAT                                                                              CGTTA                                                                              TTCAG                                         Met                                                                              Gln                                                                              Ile                                                                              Glu                                                                  ATG                                                                              CAA                                                                              ATA                                                                              GAG CT                                                                             3'                                                              TAC                                                                              GTT                                                                              TAT                                                                              CSacI                                                                              5'                                                              __________________________________________________________________________

This ligated plasmid was subsequently digested with SacI to removeexcess linkers and with SalI to provide a SalI overhang.

Fragment 1, the 2.9 K SacI fragment from pF8-102 containing the 5'coding region of human Factor VIII:C, and Fragment 2, the 6.5 KSacI-SalI fragment from pF8-6.5 which contains the 3' coding region ofthe factor, and pSV7d modified vector containing the linker adaptor wereligated together (see Truett et al, supra). This ligation mix was thenused to transform E. coli HB101, and colonies were selected byresistance to ampicillin.

Three hundred transformants were screened by colony filter hybridizationusing the BamHI-SacI 5' adaptor or the 2.9 K SacI fragment as probes.Those colonies positive with both probes were then analyzed byrestriction mapping. Plasmid pSVF8-200, which contains the entire codingregion for the human Factor VIII:C gene and a 5' untranslated regionproperly fused in transcriptional orientation to the SV40 earlypromoter, was obtained.

(E) Transfection and Culture of COS7 Cells:

The plasmids described above were transfected into COS7 cells (Guzman,Cell (1981) 23:175) using the calcium phosphate coprecipitation method(van der Eb and Graham, Meth Enzymol (1980) 65:826-39) coupled withtreatment with chloroquine diphosphate (Luthman and Magnusson, Nuc AcidsRes (1983) 11:1295-1308) using 50 μg of plasmid DNA per 5×10⁵ cells for14 hr. Cells may also be transfected by the DEAE-dextran method ofSompayrac and Danna, Proc Nat Acad Sci USA (1981) 78:7575-78.

The COS7 cells were cultured in Dulbecco's modified Eagle mediumsupplemented with 10% fetal calf serum, 100 U/mL penicillin, 100 μg/mLstreptomycin, 292 μg/mL glutamine, and 110 μg/mL sodium pyruvate.Samples were obtained from a 48-hour collection of serum-containingmedium at 88 hours post transfection.

(F) Assays

At specific intervals post transfection, medium was removed from thecells, and aliquots were stored at -70° C. Samples were tested for theirability to decrease the prolonged partial thromboplastin time of FactorVIII:C deficient plasma in a standard coagulation assay (Hardisty et al,Thromb et Diathesis Haemoloq (1962) 72:215). The more specific COATESTassay (Rosen et al, Thromb and Haemostasis (1985) 54:818-823), whichmeasures the generation of activated Factor X (Xa) as a linear functionof the concentration of exogenously supplied Factor VIII:C, was used toverify the results of the coagulation assay. The concentration ofimmunologically reactive Factor VIII:C protein in the medium wasdetermined by the application of a radioimmunoassay (RIA) developed todetect the M_(r) 92 K polypeptide and by an enzyme-linked immunosorbantassay (ELISA) specific for the M_(r) 80 K polypeptide (Nordfang et al,Thromb Haemostasis (1985) 53:346).

As shown in Table 1, expression of the M_(r) 92 K poly-peptide or of theM_(r) 80 K polypeptide alone produced no detectable activity even thoughhigh levels of each of the individual proteins were present in theconditioned media. When cells were cotransfected with pSVF8-92 andpSVF8-80 plasmids, the media contained about 20 mU/mL of coagulationactivity. The same relative level of the coagulation activity wassecreted by cells transfected with the plasmid pSVF8-200 encoding thecomplete Factor VIII:C protein.

When conditioned media from the pSVF8-92 and the pSVF8-80 singletransfectants were mixed together (using several different conditions asoutlined in Table 1) no activity was measurable.

These results indicate that a complex of the amino and carboxyl terminaldomains of Factor VIII:C retains intrinsic coagulation activity and thatthe interior domain is not essential for activity nor for the assemblyof an active complex from separate chains.

                  TABLE 1                                                         ______________________________________                                        Assay of Recombinant Factor VIII:C Activity                                   in Conditioned COS7 Cell Media                                                       Coagulation                                                                            COATEST   HC-RIA   LC-ELISA                                            Time   Activity                                                                              Activity                                                                              Assay  Assay                                  Plasmid  (sec)  mU/mL   mU/mL   mU/mL  mU/mL                                  ______________________________________                                        pSVF8-92 95.7   <0.9    <0.1    0.15   <0.0002                                pSVF8-80 97.2   <0.9    <0.1    <0.01  1.36                                   pSVF8-92 &                                                                             56.1   22.5    20.4    0.05   1.13                                   pSVF8-80.sup.a                                                                pSVF8-200                                                                              47.7   70.0    43.2    0.12   0.28                                   none     94.6   <0.9    <0.1    <0.0   <0.0002                                pSVF8-92J +                                                                            95.7   <0.9    <0.1    --                                            pSVF8-80.sup.b†                                                        ______________________________________                                         .sup.a plasmids were cotransfected into the same cells                        .sup.b plasmids were transfected into separate cells, and the supernatant     mixed 48 hours later                                                          .sup.† A variety of mixing conditions were tested, including           preincubation for various times up to 2 hr at 37° C., 20°       C., or 4° C. in the presence or absence of 10 mM CaCl.sub.2. The       value reported in this table is representative of the data obtained.     

In Table 1, Coagulation Time and Activity were obtained as follows:Aliquots of 75 μL of media, conditioned by the growth of COS7 cellstransfected with the indicated plasmids or mock transfected, wereassayed for their ability to decrease the prolonged partialthromboplastin time of Factor VIII:C-deficient plasma in the one-stageassay. Briefly, 75 μL of Platelin (General Diagnostics) was incubatedfor 3 min at 37° C., followed by the addition of 75 μL of FactorVIII:C-deficient plasma plus 75 μL of the test sample for an additional5 min incubation at 37° C. A 75 μL aliquot of prewarmed 0.025 M CaCl₂was added, and the clotting time measured with a Becton-Dickinsonfibrometer. Normal human plasma diluted in COS7 cell medium was used asa standard. One mU of activity is assumed to correspond to approximately100 pg of Factor VIII:C protein (Fay et al, Proc Nat Acad Sci USA (1982)79:7200).

In Table 1, the COATEST assay (Kabi) was used to measure the generationof activated Factor X (Xa) as a linear function of the concentration ofFactor VIII:C. The concentration of Factor Xa is measured by theproteolytic cleavage of the chromogen para-nitroaniline from a syntheticpeptide substrate for Factor Xa. Normal human plasma diluted in 50 mMTris-HCl, pH 7.3, 0.2% BSA was used as the standard.

For the RIA assay in Table 1, purified canine Factor VIII:C-inhibitoryIgG was coated onto the wells of a 96-well polystyrene microtiter plateat a concentration of 3.5 μg/mL in 0.1M sodium carbonate buffer, pH 9.8,by overnight incubation at 37° C. The plates were washed 3 times with0.1M NaCl, 0.05% Tween® 20 followed by incubation with a mixture of testmedium samples and iodinated FVIII:C M_(r) 92 K protein, both diluted in0.05M imidazole, 0.1M NaCl, 1% bovine serum albumin, 0.05% Tween® 20, pH7.3. The FVIII:C M_(r) 92 K protein was isolated from plasma and wasgreater than 50% homogeneous as estimated by SDS-PAGE and silverstaining. After incubation for 16 hr at room temperature, the plateswere washed, and the amount of ¹²⁵ I in the individual wells wasmeasured in a gamma counter. An intermediate purified commercial FactorVIII:C preparation (Factor VIII, NORDISK) with a specific activity of0.5 unit of coagulation activity per mg was used as the standard. Thisstandard was calibrated against the World Health Organization ThirdInternational Factor VIII:C standard. We defined our intermediatepurified standard to contain a M_(r) 92 K RIA activity/Factor VIII:Ccoagulation activity ratio of 1.

For the ELISA assay in Table 1, purified human Factor VIII:C-inhibitoryIgG was coated onto the wells of a 96-well PVC microtiter plate at aconcentration 4.5 μg/mL in 0.1M sodium carbonate, pH 9.8, by overnightincubation at 37° C. The wells were washed as above andperoxidase-conjugated F(ab')₂ fragments of the human inhibitory IgGdiluted in 0.1M imidazole, 0.15M NaCl, 1% BSA, 0.05% Tween® 20, pH 7.3,were added for a final incubation of 16 hr at room temperature. Thecolor was developed with o-phenylenediamine solution. Normal human serumwas used as a standard.

To verify that the observed coagulation activity was due to FactorVIII:C, the sensitivity of the coagulation to inhibition by antibodyspecific for Factor VIII:C was determined. Prior to assay, aliquots ofconditioned media were pre-incubated for 2 hr at 37° C. in the presenceof dilutions of normal human serum or of serum from a hemophiliac whohad developed a high titer of inhibitory antibodies to Factor VIII:C. Asshown in Table 2, the activity of the complete molecule, as well as thatof the M_(r) 92 K-80 K complex was reduced specifically by theinhibitory serum. The same results were obtained using three differentinhibitory monoclonal antibodies which bind to the M_(r) 80 K species.Inhibition of Factor VIII:C activity using inhibitory serum was studiedas follows: 160 μL of the indicated COS7 cell conditioned medium wereincubated with 20 μL of a 100-fold dilution of human Factor VIII:Cinhibitory serum (Bethesda titer 1500 units) or a similar dilution ofpooled normal human serum, or buffer alone (50 mM imidazole, 0.1M NaCl,100 μg/mL BSA pH 7.3) for 2 hr at 37° C. These samples were then assayedfor residual coagulation activity as outlined above.

                  TABLE 2                                                         ______________________________________                                        Coagulation Inhibition Assay                                                                           Coagulation Time                                     Plasmid          serum   (secs)                                               ______________________________________                                        pSVF8-80 + pSVF8-92                                                                            Normal  51.9                                                                  Immune  74.5                                                                  Buffer  54.4                                                 pSVF8-200        Normal  46.4                                                                  Immune  69.4                                                                  Buffer  46.8                                                 ______________________________________                                    

The inhibition experiment was repeated using monoclonal antibodies, asfollows: 100 μL of conditioned medium were incubated for 2 hr at 37° C.with either 10 μL of a 1 μg/μL solution of anti-Factor VIII:C monoclonalantibody from Hybritech (Bethesda titer 14,000 units) or buffer, andthen assayed as above. The results are shown in Table 3.

                  TABLE 3                                                         ______________________________________                                        Coagulation Inhibition Assay                                                                           Coagulation Time                                     Plasmid          serum   (secs)                                               ______________________________________                                        pSVF8-92 + pSVF8-80                                                                            Immune  72.9                                                                  Buffer  48.0                                                 pSVF8-200        Immune  60.9                                                                  Buffer  44.9                                                 ______________________________________                                    

To demonstrate more clearly the existence of a two chain complex, theactive species was partially purified from the COS7 cell media bypassage over a MAb column specific for the M_(r) 80 K portion. As shownin Table 4, approximately 65% of the applied activity was retained bythe column and 50% of this bound material was eluted in an active formand at a fivefold greater concentration than in the initial media. Thusan active complex can be isolated by affinity chromatography using anantibody specific for only the M_(r) 80 K species. 100 μg of an anti-80K monoclonal antibody (56 IgG) (Nordfang et al, Thromb Haemostasis(1985) 53:346) coupled to Sepharose® CL4B were incubated overnight at20°C. with 1.4 mL of medium containing a total of 6.2 mU of activity(measured by the COATEST Assay obtained from COS7 cells cotransfectedwith pSVF8-92 and pSVF8-80 plasmids). After incubation, the slurry wasloaded into a column and the flowthrough fraction was collected. Thecolumn was washed with 300 μL of Buffer A (50 mM imidazole, 0.1M NaCl,0.1% sodium insulin, 0.2% NaN₃, pH 7.3) and then eluted with 300 μL ofBuffer B (2.5M NaCl, 50% ethylene glycol, 0.5M imidazole, 0.1M CaCl₂,0.1% sodium insulin, 0.2% NaN₃, pH 7.3).

                  TABLE 4                                                         ______________________________________                                        Partial Purification of M.sub.r 92 K-80 K Coagulation                         Active Complex                                                                              COATEST   80 K ELISA                                            Fraction      U/mL      U/mL                                                  ______________________________________                                        Media         .0044     0.175                                                 Flowthrough   .0017     0.13                                                  Eluate        .0200     0.76                                                  ______________________________________                                    

Results reported here demonstrate that expression of the linker ("B")region, containing 918 amino acids or about 40% of the total for theintact protein, is not required for Factor VIII:C activity.Co-expression of individual M_(r) 92 K and M_(r) 80 K regions results ina level of Factor VIII:C activity comparable to that obtained from theexpression of the whole Factor VIII:C coding region. These proteinsassemble in vivo to form an active complex linked by a calcium bridge.The assembly does not require the presence of the B region and occursefficiently for the two chains expressed in trans.

It is evident from the above results that Factor VIII:C activity can beachieved by directly producing an N-terminal fragment and a C-terminalfragment which are independently expressed, each having its own signalsequence. Thus, Factor VIII:C can be obtained more efficiently, sincethe large precursor need not be cloned and used as the coding sequencefor the Factor VIII:C activity. Thus, cells may be employed forexpression of Factor VIII:C which may be deficient in the capability forproper maturation of the full-length Factor VIII:C protein.

Example 2

Expression of the M_(r) 92 K protein in COS7 cells using the pSVF8-92construction was low compared to the amount of M_(r) 80 K proteinproduced. The M_(r) 92 K protein is apparently retained and/or degradedin the Golgi pathway, and is not efficiently processed or exported.Accordingly, the construction was modified in an attempt to increase thelevel of M_(r) 92 K protein. Modifications of the following types weremade: Changes in the 5' untranslated sequence of the Factor VIII:C gene;inclusion of heterologous 5' untranslated and leader sequences; andchanges in the 3' untranslated sequences. These constructs aresummarized below.

(A) 5' Untranslated Region Modifications

Plasmid pSVF8-92B. This plasmid is a derivative of pSVF8-92 in which the30 bp of 5' untranslated sequence of pSVF8-92 is replaced with theentire 5' untranslated region of human Factor VIII:C cDNA (nucleotides 1to 171; see FIG. 8 of Truett et al, supra), with a deletion of the G-Ctails (by in vitro site-specific mutagenesis), and the three basechanges shown below at the starting ATG (at position +172, FIG. 8,Truett et al, supra) to conform to Kozak's preferred sequences forefficient message translation in eukaryotic cells:

Factor VIII:C: GTCATG CAA

Kozak consensus: ACCATG G

This change alters the second amino acid of the signal peptide to Glufrom Gln.

Plasmid pSVF8-92E. This plasmid is a derivative of pSVF8-92B in whichthe polylinker derived from pSV7d 5' to the Factor VIII:C sequences isremoved with the exception of the SalI site, and the ATG codon in the 5'untranslated region (at 41 according to Truett et al, supra) is alteredto ATT, by in vitro mutagenesis.

(B) Addition of Heterologous 5' Sequences

Plasmids pSVF8-92G, H, and I. These plasmids are derivatives ofpSVF8-92B in which the 5' untranslated region as well as the naturalFactor VIII:C signal sequences are replaced with the analogous regionfrom the human tissue plasminogen activator (tPA) cDNA. In pSVF8-92G thefirst 35 amino acids (signal and pro-sequences) of the tPA pre-proregion are joined to mature Factor VIII:C M_(r) 92 K with a serinesubstituted for the first amino acid (alanine) of the M_(r) 92 Kprotein. In pSVF8-92H the first 32 amino acids of the tPA pre-pro regionare joined to mature Factor VIII:C M_(r) 92 K protein. In pSVF8-92I, thefirst 23 amino acids of the tPA pre-pro region are joined to matureFactor VIII:C M_(r) 92 K protein. The tPA sequences are the same asthose described for pSVF8-80.

Plasmid pSVF8-92J. This plasmid is a derivative of pSVF8-92G in whichthe tPA 5' region is replaced with 75 bp of Herpes simplex virus-1(HSV-1) gD 5' untranslated sequences and 75 bp of HSV-1 gD signalsequence. pSVF8-92J also lacks the Ala → Ser substitution (R. J. Watsonet al, Science (1982) 218:381-384).

(C) 3' Untranslated Region Changes

Plasmid pSVF8-92C. This plasmid is a variation of pSVF8-92B in which theM_(r) 92 K coding region is fused directly to the translational stopcodon and natural 3' untranslated sequences of human Factor VIII:C cDNA.

Plasmid pSVF8-92L. This plasmid is a derivative of pSVF8-92C in whichthe 3' untranslated region of pSVF8-92C is replaced with the 3'untranslated region of pSVF8-80.

(D) Results

Each of the plasmids of parts A-C above was transfected into COS7 cellsalong with pSVF8-80 as described in Example 1 and the media tested forFactor VIII:C activity as in Example 1(F).

Plasmid pSVF8-92B, the first tested, showed activity levels ranging from2-to-8-fold better than pSVF8-92. Of the remaining plasmids pSVF8-92Eappeared to be the best, being 1.65-fold better than pSVF8-92B.pSVF8-92J and I also produced substantially higher expression levelsthan pSVF8-92, being close to that of pSVF8-92E. The expression level ofpSVF8-92G approximated that of pSVF8-92, whereas that of pSVF8-92H wassubstantially less than pSVF8-92. The expression levels of bothpSVF8-92C and pSVF8-92L appear to be equivalent to that of pSVF8-92E.

Example 3

This example describes the preparation of constructs for producingpolypeptides that consist of the M_(r) 92 K chain and a portion of the Bdomain. These derivatives were made in an attempt to develop a heavychain that is more stable and/or assembles more efficiently into anactive complex with the light chain. The derivatives were chosen tomimic molecular species that have been observed in plasma-derivedpreparations of Factor VIII:C and in cell lysates and conditioned mediafrom cells expressing recombinant full-length Factor VIII:C.Polypeptides of approximately the same size could possibly arise bythrombin cleavages of full-length Factor VIII:C.

(A) pSVF8-92S: This plasmid encodes a 982 amino acid heavy chain and wasprepared from a full-length cDNA plasmid pSVF8-302 by cleavage at thefirst SacI site of the B-domain coding region. An oligonucleotideadaptor was used to install a translational stop codon and fuse thecoding sequence to the natural human Factor VIII:C 3' untranslatedsequence beginning at the first BalI site. This plasmid encodes thefirst 978 amino acids of native human Factor VIII:C and 4 substitutedamino acid residues at the carboxy terminus.

(B) pSVF8-160: This plasmid provides a 1323 amino acid heavy chain andwas prepared from a full-length clone (designated pSVF8-303) similar topSVF8-200, but having the 5' untranslated region of pSVF8-92E. pSVF8-303was cleaved with EcoRV and SmaI, and the blunt ends were ligatedtogether to form pSVF8-160. This plasmid encodes the first 1315 aminoacids of Factor VIII:C. Eight substituted amino acids are added at thecarboxyl terminus as a result of the fusion of the polylinker of thevector pSV7d.

(C) pSVF8-170: This plasmid provides a 1416 amino acid heavy chain andwas also prepared from pSVF8-303. pSVF8-303 was partially digested withBglII, and the resulting 6811 bp fragment was gel isolated and the endsligated together to form pSVF8-170. This plasmid encodes the first 1405amino acids of Factor VIII:C and has a carboxyl extension of 11 aminoacids due to fusion of the polylinker of the vector pSV7d.

(D) PSVF8-120: This plasmid provides a 1107 amino acid heavy chain andwas prepared from pSVF8-303. The plasmid pSVF8-303 was digested withApaI and the cohesive ends were filled in with T4 polymerase. Theresulting molecule was further digested with SmaI, the DNA self-ligatedand propagated in E. coli HB101. This plasmid encodes 1102 amino acidsfrom the amino terminus of Factor VIII:C plus an additional 5 aminoacids at the carboxyl terminus, encoded by the pSV7d polylinker.

(E) Results

Each of the plasmids of parts A-D was transfected into COS7 cells alongwith pSVF8-80, and the media tested for Factor VIII:C activity, asdescribed in Example 1.

All of these plasmids showed substantially reduced expression levelscompared to that of pSVF8-92E. Interestingly, though, the ratio of RIAto COATEST activity for pSVF8-160 and pSVF8-170 is about 1.8, comparedto 7.2 for pSVF8-92E. This result suggests that these longer heavy chainderivatives have a higher specific activity, that is, they are moreefficiently assembled into active subunit complexes than the M_(r) 92 Kmolecule itself. Also, the ratio of coagulation activity to COATESTactivity is lower for the longer heavy chains at about 1.7 compared to2.3 for M_(r) 92 K and 1.35 for the complete molecule, suggesting thatthese longer polypeptides form complexes which are not as activated asthat of the M_(r) 92 K +M_(r) 80 K complex.

Example 4

This example describes the preparation of stable CHO cell lines thatproduce the Factor VIII:C M_(r) 92 K-80 K chain complex.

(A) Preparation of a plasmid encoding a selectable marker

The plasmid pAd-DHFR, bearing the murine DHFR cDNA, was constructed byfusing the major late promoter from adeno-virus-2 (Ad-MLP, map units16-27.3) to the 5' untranslated sequences of the mouse DHFR CDNA (J.H.Nunberg et al, Cell (1980) 19:355-64). SV40 DNA encoding part of theearly transcription unit, including the intron of the small t antigengene, and having the SV40 early region transcriptional terminationregion, was obtained from pSV2-neo (Southern and Berg, J Mol Appl Gen(1982) 1:327-41) and fused to the 3' untranslated end of the DHFR CDNA.These three segments were subcloned into pBR322 to obtain plasmidpAd-DHFR.

(B) Transfection and culture of CHO cells

CHO-DUKX-B11 cells carrying non-functional genes for dihydrofolatereductase (Urlaub and Chasin, Proc Nat Acad Sci USA (1980) 77:4216-4220)were transfected with a calcium phosphate coprecipitate of threeplasmids: pSVF8-92C, pSVF8-92E, or pSVF8-80, and pAd-DHFR following themethod of Graham and Van der Eb, supra, and modifications described byWigler et al, Cell (1978) 14:725-731 and Lewis et al, Somatic Cell Genet(1980) 6:333-347. Coprecipitates contained up to 10 μg of each plasmid.Cells were selected for expression of the DHFR (positive) phenotype in amedium deficient in hypoxanthine and thymidine.

After isolation of DHFR positive clones and identification of thoseproducing Factor VIII:C activity, the resulting cell lines were grown inmethotrexate to amplify the DHFR genes and coamplify the Factor VIII:Cgenes. This selection was performed by plating cells in mediumcontaining methotrexate in concentrations ranging from 0.025 to 0.2 μM.Methotrexate resistant clones were again assayed for Factor VIII:Cactivity.

(C) Assay Methods

Conditioned media from these DHFR positive clones were assayed by ELISAfor Factor VIII:C light chain immunoreactivity by the method of Nordfanget al, Thromb Haemostas (1985) 53:346-50. Factor VIII:C heavy chainimmunoreactivity was evaluated using a radioimmunoassay (RIA) describedby R. L. Burke et al, J Biol Chem (1986) 261:12574-78. Active FactorVIII:c complexes formed by co-expression of the 92K and 80K M_(r)glycoproteins were measured using the COATEST assay described in Example1.

(D) CHO lines expressing active 92K-80K M_(r) complexes

Shown in Table 5 are four independent CHO cell lines that simultaneouslyexpress products of all three plasmids used for transfection. The FactorVIII:C activity values shown in Table 5 are those initially observed.Expression of glycoproteins by stable cell lines usually improves afterpassage in T-75 flask cultures. An example of this can be seen for theline 10-C2, which ultimately produced 200 mU Factor VIII:C activity permL conditioned medium (Table 6). Cloning these stable cell linesillustrates that the independently expressed heavy and light chains ofFactor VIII:C can assemble into an active complex and be secreted byChinese hamster ovary cells.

                  TABLE 5                                                         ______________________________________                                        CHO cell lines producing active 92 K-80 K complexes                           Clone  Transfected DNA     mU COATEST/mL                                      ______________________________________                                        11-D6  pSVF8-92C, pSVF8-80, pAd-DHFR                                                                     43                                                 11-D5  pSVF8-92C, pSVF8-80, pAd-DHFR                                                                     30                                                 8-C1   pSVF8-92E, pSVF8-80, pAd-DHFR                                                                     18.2                                               10-C2  pSVF8-92E, pSVF8-80, pAd-DHFR                                                                     70.0                                               ______________________________________                                    

That the three plasmids were integrated into the chromosomes of the CHOcells is suggested by the fact that the cell lines of Table 5 could begrown for many passages without loss of Factor VIII:C expression. It wasthen necessary to determine if expression of Factor VIII:C glycoproteinscould be co-amplified by methotrexate selection. All four of these celllines were placed under selection in several concentrations ofmethotrexate. Resistant colonies (DHFR genes amplified) were obtainedfor each line and these were screened for Factor VIII:C activity.Expression of Factor VIII:C was lost or unchanged in methotrexateresistant 11-D5 and 11-D6 clones. Expression of Factor VIII:C variedamong methotrexate resistant clones derived from 10-C2 and 8-C1 (shownin Table 6).

Twenty-two methotrexate-resistant 8-Cl clones were examined, the datafor 10 of which are reported in Table 6. The amount of Factor VIII:Camplification varies among clones, suggesting that either one of thesubunit genes may have been co-amplified with the DHFR cassette, or bothof them, or neither one. Note clones 8C1-A2, 8C1-C2, and 8C1-C5 asexamples of these four possibilities. Similarly, 30methotrexate-selected derivatives of 10-C2 were evaluated, the data for20 of which are also represented in Table 6. These also contain aspectrum of activity. Note clones 10C2-A2, 10C2-D2, 10C2-B5, and 10C2-C6as examples of the four different co-amplification possibilities.

                  TABLE 6                                                         ______________________________________                                                conc. MTX COATEST    LC-ELISA                                                                              HC-RIA                                   Clone   (μM)   (mU/mL)    (mU/mL) (mU/mL)                                  ______________________________________                                        8-C1    0         18         1275    n.d.                                     8C1-A1  0.1       <50        1750    80                                       8C1-A2  0.1       60         1950    >1000                                    8C1-A5  0.05      2          100     10                                       8C1-B3  0.025     33         1950    1000                                     8C1-B4  0.025     50         3550    820                                      8C1-B5  0.025     35         1950    >1000                                    8C1-C2  0.025     130        13,100  >>1000                                   8C1-C3  0.025     165        3900    >>>1000                                  8C1-C5  0.025     30         1750    760                                      10-C2   0         200        1400    700                                      10C2-A1 0.05      61         1600    400                                      10C2-A2 0.1       67         6700    700                                      10C2-A4 0.05      63         2250    1200                                     10C2-A5 0.05      183        9450    2660                                     10C2-A6 0.05      320        8600    7400                                     10C2-B1 0.05      408        8100    4300                                     10C2-B3 0.05      134        800     9800                                     10C2-B4 0.05      394        18,000  7800                                     10C2-B5 0.05      461        15,000  8400                                     10C2-B6 0.05      247        2200    9800                                     10C2-C1 0.1       160        8100    7600                                     10C2-C2 0.05      228        6000    5600                                     10C2-C3 0.05      294        14,850  2650                                     10C2-C5 0.05      294        12,400  5400                                     10C2-C6 0.05      100        1350    520                                      10C2-D2 0.05      496        1560    16,400                                   10C2-D3 0.05      242        10,200  2260                                     10C2-D4 0.05      165        14,100  3500                                     10C2-D5 0.05      316        7800    5200                                     10C2-D6 0.05      141        1600    6400                                     ______________________________________                                    

Among the CHO lines described in Table 6 is one (10C2-D2) that produces0.5 U/mL of active Factor VIII:C complex, which is one half theconcentration found in normal human plasma. For analysis andpurification of Factor VIII:C material, CHO cell lines expressing FactorVIII:C polypeptides were grown in laboratory scale fermentations toproduce 1-2 liter quantities of tissue culture fluid. Assay of thismaterial showed that approximately 10% to 20% of immunoreactive FactorVIII:C from unamplified lines is active in the COATEST. In amplifiedlines, the percentage of active material drops to 2% to 5% of the totalimmunoreactive product. This means that only a fraction of the heavy andlight chains of FVIII:C is assembled into active complexes. Theremainder may exist as free subunits or in degraded forms.

Plasmids pSVF8-92 and pSVF8-80 were deposited at the American TypeCulture Collection (ATCC) on 24 Jan. 1986 and given ATCC Accession Nos.40222 and 40223, respectively. Plasmid pSVF8-200 was deposited at theATCC on 17 Jul. 1985 and was given ATCC Accession No. 40190.

Example 5

This example describes modification of the plasmid pSVF8-80 to correctthe amino terminal amino acid of the FVIII:C light chain glycoprotein. Aconsequence of engineering, which provided the signal peptide needed forindependent secretion of the 80 K M_(r) glycoprotein (Example 1) is thesubstitution of Ser for the normal aminoterminal residue of human plasmaFVIII:C light chains. New plasmids were made in an attempt to change thetPA pre-pro peptide sequence, so that the FVIII:C light chain will havethe Glu residue at its amino terminus instead of the mutant Ser residueafter proteolytic processing.

The FVIII:C light chain is thought to be cleaved from the full-lengthFVIII:C precursor before secretion, i.e. intracellularly, by a proteaseresident in the Golgi apparatus. This cleavage occurs between amino acidresidues 1648 and 1649 (Arg-Glu). On polyacrylamide gels the lightchains appear as a doublet of 77 and 80 K M_(r) bands, representingpolypeptides having one or two N-linked oligosaccharides. Independentsecretion of light chains was achieved by fusion of the light chaincoding region of the FVIII:C cDNA to the cDNA of tPA. In the process ofsupplying the tPA signal peptide, however, the amino terminus of theFVIII:C light chain was mutated from the native glutamic acid residue toa serine. Although this mutant recombinant light chain displaysmolecular characteristics similar to the chain derived from full-lengthrecombinant FVIII:C, there is preliminary evidence that 1) it may not bealternatively glycosylated in the same manner as the chain cleaved fromthe FVIII:C precursor, 2) it may behave differently during purificationby ion exchange and vWF Sepharose® chromatography, and 3) it may bedifferent antigenically from authentic light chain.

The tPA pre-pro peptide sequence requires three proteolytic cleavages torelease the mature polypeptide. Shown below is the translation of theprotein coding sequence of pSVF8-80 in the region of the tPA-FVIII:C 80K fusion:

    __________________________________________________________________________    pSVF8-80:                                                                     __________________________________________________________________________    -35            -30            -25                                             Met                                                                              Asp                                                                              Ala                                                                              Met                                                                              Lys                                                                              Arg                                                                              Gly                                                                              Leu                                                                              Cys                                                                              Cys                                                                              Val                                                                              Leu                                                                              Leu                                                                              Leu                                    ATG                                                                              GAT                                                                              GCA                                                                              ATG                                                                              AAG                                                                              AGA                                                                              GGG                                                                              CTC                                                                              TGC                                                                              TGC                                                                              TGT                                                                              GTG                                                                              CTG                                                                              CTG                                       -20            -15   *        -10   *                                      Cys                                                                              Gly                                                                              Ala                                                                              Val                                                                              Phe                                                                              Val                                                                              Ser                                                                              Pro                                                                              Ser                                                                              Gln                                                                              Glu                                                                              Ile                                                                              His                                                                              Ala                                    TGT                                                                              GGA                                                                              GCA                                                                              GTC                                                                              TTC                                                                              GTT                                                                              TCG                                                                              CCC                                                                              AGC                                                                              CAG                                                                              GAA                                                                              ATC                                                                              CAT                                                                              GCC                                          -5 @           1           5                                            Arg                                                                              Phe                                                                              Arg                                                                              Arg                                                                              Gly                                                                              Ala                                                                              Arg                                                                              Ser                                                                              Ile                                                                              Thr                                                                              Arg                                                                              Thr                                                                              Thr                                                                              Leu                                    CGA                                                                              TTC                                                                              AGA                                                                              AGA                                                                              GGA                                                                              GCC                                                                              AGA                                                                              TCT                                                                              ATA                                                                              ACT                                                                              CGT                                                                              ACT                                                                              CTT                                                                              CAG                                          10                                                                      Gln                                                                              Ser                                                                              Asp                                                                     CAG                                                                              TCT                                                                              GAT                                                                     __________________________________________________________________________

The signal peptidase cleavage has been thought to occur on the carboxyside of either Ser (position -13) or Ala (position -8), indicated byasterisks. The second cleavage probably occurs on the carboxy side ofArg (position -4, indicated by @ above). The third processing event isproteolysis at the Arg-Ser bond to release a Gly-Ala-Arg tripeptide andleave a Ser (position 1) aminoterminus on the mature tPA or FVIII:Clight chain poly-peptides.

(A) Preparation of plasmids

(1) pSVF9-8OKG:

The Ser codon (position 1) was changed by site-directed mutagenesis to aGlu codon (position 1). This was done in an effort to allow the firsttwo proteolytic processing events to occur normally, and test whetherthe Arg-Glu protease could recognize and cleave the dipeptide in analtered context, i.e., where the tPA tripeptide is substituted for theFVIII:C B domain. The tPA-80 K chain fusion region is shown below.Otherwise, this plasmid is identical to pSVF8-80.

    __________________________________________________________________________    pSVF8-80KG:                                                                   __________________________________________________________________________    -35            -30            -25                                             Met                                                                              Asp                                                                              Ala                                                                              Met                                                                              Lys                                                                              Arg                                                                              Gly                                                                              Leu                                                                              Cys                                                                              Cys                                                                              Val                                                                              Leu                                                                              Leu                                                                              Leu                                    ATG                                                                              GAT                                                                              GCA                                                                              ATG                                                                              AAG                                                                              AGA                                                                              GGG                                                                              CTC                                                                              TGC                                                                              TGC                                                                              TGT                                                                              GTG                                                                              CTG                                                                              CTG                                       -20            -15   *        -10   *                                      Cys                                                                              Gly                                                                              Ala                                                                              Val                                                                              Phe                                                                              Val                                                                              Ser                                                                              Pro                                                                              Ser                                                                              Gln                                                                              Glu                                                                              Ile                                                                              His                                                                              Ala                                    TGT                                                                              GGA                                                                              GCA                                                                              GTC                                                                              TTC                                                                              GTT                                                                              TCG                                                                              CCC                                                                              AGC                                                                              CAG                                                                              GAA                                                                              ATC                                                                              CAT                                                                              GCC                                          -5 @           1           5                                            Arg                                                                              Phe                                                                              Arg                                                                              Arg                                                                              Gly                                                                              Ala                                                                              Arg                                                                              Glu                                                                              Ile                                                                              Thr                                                                              Arg                                                                              Thr                                                                              Thr                                                                              Leu                                    CGA                                                                              TTC                                                                              AGA                                                                              AGA                                                                              GGA                                                                              GCC                                                                              AGA                                                                              GAA                                                                              ATA                                                                              ACT                                                                              CGT                                                                              ACT                                                                              CTT                                                                              CAG                                          10                                                                      Gln                                                                              Ser                                                                              Asp                                                                     CAG                                                                              TCT                                                                              GAT                                                                     __________________________________________________________________________

(2) pSVF8-80S:

Twelve codons were deleted from pSVF8-80 by in vitro mutagenesis, andthe Ser (position 1) codon changed to a codon for Glu. This placed theGlu FVIII:C light chain residue after Ser 23 of the putative tPA signalpeptide (indicated by an asterisk). Cleavage by signal peptidase on thecarboxy side of Ser 23 releases the non-mutant FVIII:C light chain. ThetPA -80K chain fusion region of pSVF8-80S is shown below. Otherwise thisplasmid is identical to pSVF8-80.

    __________________________________________________________________________    pSVF8-80S:                                                                    __________________________________________________________________________    -23      -20            -15            -10                                    Met                                                                              Asp                                                                              Ala                                                                              Met                                                                              Lys                                                                              Arg                                                                              Gly                                                                              Leu                                                                              Cys                                                                              Cys                                                                              Val                                                                              Leu                                                                              Leu                                                                              Leu                                    ATG                                                                              GAT                                                                              GCA                                                                              ATG                                                                              AAG                                                                              AGA                                                                              GGG                                                                              CTC                                                                              TGC                                                                              TGC                                                                              TGT                                                                              GTG                                                                              CTG                                                                              CTG                                                -5          *  1           5                                      Cys                                                                              Gly                                                                              Ala                                                                              Val                                                                              Phe                                                                              Val                                                                              Ser                                                                              Pro                                                                              Ser                                                                              Glu                                                                              Ile                                                                              Thr                                                                              Arg                                                                              Thr                                    TGT                                                                              GGA                                                                              GCA                                                                              GTC                                                                              TTC                                                                              GTT                                                                              TCG                                                                              CCC                                                                              AGC                                                                              GAG                                                                              ATA                                                                              ACT                                                                              CGT                                                                              ACT                                                10                                                                Thr                                                                              Leu                                                                              Gln                                                                              Ser                                                                              Asp                                                                              Gln                                                                              Glu                                                                              Glu                                                                              Ile                                                                              Asp                                                                              Tyr                                                                              Asp                                                                              Asp                                                                              Thr                                    CTT                                                                              CAG                                                                              CAG                                                                              TCT                                                                              GAT                                                                              CAA                                                                              GAG                                                                              GAA                                                                              ATT                                                                              GAC                                                                              TAT                                                                              GAT                                                                              GAT                                                                              ACC                                    __________________________________________________________________________

(3) pSVF8-80R:

A deletion of three codons of pSVF8-80, to remove the tPApro-tripeptide, was made by in vitro mutagenesis, and the Ser(position 1) codon was changed to one for Glu. This places a Glu residueafter Arg32 of the tPA pro-peptide, marked with @ on the tPA-80 K chainfusion region of pSVF8-80R shown below:

    __________________________________________________________________________    pSVF8-80R:                                                                    __________________________________________________________________________    -32   -30            -25            -20                                       Met                                                                              Asp                                                                              Ala                                                                              Met                                                                              Lys                                                                              Arg                                                                              Gly                                                                              Leu                                                                              Cys                                                                              Cys                                                                              Val                                                                              Leu                                                                              Leu                                                                              Leu                                    ATG                                                                              GAT                                                                              GCA                                                                              ATG                                                                              AAG                                                                              AGA                                                                              GGG                                                                              CTC                                                                              TGC                                                                              TGC                                                                              TGT                                                                              GTG                                                                              CTG                                                                              CTG                                             -15            -10            -5                                     Cys                                                                              Gly                                                                              Ala                                                                              Val                                                                              Phe                                                                              Val                                                                              Ser                                                                              Pro                                                                              Ser                                                                              Gln                                                                              Glu                                                                              Ile                                                                              His                                                                              Ala                                    TGT                                                                              GGA                                                                              GCA                                                                              GTC                                                                              TTC                                                                              GTT                                                                              TCG                                                                              CCC                                                                              AGC                                                                              CAG                                                                              GAA                                                                              ATC                                                                              CAT                                                                              GCC                                             @  1                          10                                     Arg                                                                              Phe                                                                              Arg                                                                              Arg                                                                              Glu                                                                              Ile                                                                              Thr                                                                              Arg                                                                              Thr                                                                              Thr                                                                              Leu                                                                              Gln                                                                              Ser                                                                              Asp                                    CGA                                                                              TTC                                                                              AGA                                                                              AGA                                                                              GAG                                                                              ATA                                                                              ACT                                                                              CGT                                                                              ACT                                                                              CTT                                                                              CAG                                                                              CAG                                                                              TCT                                                                              GAT                                    __________________________________________________________________________

This construction was made in the hope that cleavage by a Golgi-residentprotease with dibasic specificity would release FVIII:C light chainshaving Glu amino termini.

(4) pSVF8-80A:

Seven codons of pSVF8-80 were deleted by sitedirected mutagenesis,removing the DNA encoding the putative tPA pro sequence, and the Ser(position 1) codon was replaced by a Glu codon after codon 28 (Ala) ofthe putative tPA signal peptide coding sequence (indicated by anasterisk below). Cleavage by signal peptidase on the carboxy side ofAla₂₈ will release non-mutant FVIII:C light chain. The tPA-80K chainfusion region is shown below. Otherwise, this plasmid is identical topSVF8-80.

    __________________________________________________________________________    pSVF8-80A:                                                                    __________________________________________________________________________    -28      -25            -20            -15                                    Met                                                                              Asp                                                                              Ala                                                                              Met                                                                              Lys                                                                              Arg                                                                              Gly                                                                              Leu                                                                              Cys                                                                              Cys                                                                              Val                                                                              Leu                                                                              Leu                                                                              Leu                                    ATG                                                                              GAT                                                                              GCA                                                                              ATG                                                                              AAG                                                                              AGA                                                                              GGG                                                                              CTC                                                                              TGC                                                                              TGC                                                                              TGT                                                                              GTG                                                                              CTG                                                                              CTG                                                -10            -5          *                                      Cys                                                                              Gly                                                                              Ala                                                                              Val                                                                              Phe                                                                              Val                                                                              Ser                                                                              Pro                                                                              Ser                                                                              Gln                                                                              Glu                                                                              Ile                                                                              His                                                                              Ala                                    TGT                                                                              GGA                                                                              GCA                                                                              GTC                                                                              TTC                                                                              GTT                                                                              TCG                                                                              CCC                                                                              AGC                                                                              CAG                                                                              GAA                                                                              ATC                                                                              CAT                                                                              GCC                                    1                          10                                                 Glu                                                                              Ile                                                                              Thr                                                                              Arg                                                                              Thr                                                                              Thr                                                                              Leu                                                                              Gln                                                                              Ser                                                                              Asp                                                                              Gln                                                                              Glu                                                                              Glu                                                                              Ile                                    GAG                                                                              ATA                                                                              ACT                                                                              CGT                                                                              ACT                                                                              CTT                                                                              CAG                                                                              CAG                                                                              TCT                                                                              GAT                                                                              CAA                                                                              GAG                                                                              GAA                                                                              ATT                                    __________________________________________________________________________

(B) Expression and protein sequence analysis

(1) Transfection into COS7 Cells:

COS7 cells were transfected using the DEAE-dextran procedure describedin Example 1, and conditioned media were assayed by the LC-ELISA. Allfour derivatives of pSVF8-80 encode 80 K M_(r) glycoproteins that arereactive in the LC-ELISA and that can be immunoprecipitated afterbiosynthetic radio-labeling with various anti-FVIII:C light chainantibodies. Except for pSVF8-80R, all the derivatives lead to secretionof about the same amount of 80K glycoprotein as pSVF8-80. Secretion of80K glycoprotein from cells transfected with pSVF8-80R is very poor,usually less than 25% of that produced from the other plasmids. Inaddition, the appearance of this FVIII:C light chain is different on gelelectrophoresis, where the bands are always diffuse.

(2) Expression in CHO Cells:

Each of these plasmids was introduced into DUKX-B11 CHO cells withpAd-DHFR as described in Example 4. Permanent cell lines wereestablished for production of each type of light chain. Expression ofthe 80K M_(r) glycoproteins in CHO cells is very similar to expressionin COS7 cells, with respect to the amounts of glycoprotein secreted andthe appearance of the 80K bands on gel electrophoresis. CHO linestransfected with pSVF8-80R produced such a low level of 80K glycoproteinthat analysis of this material was not done.

3. Purification and amino acid sequence analysis

Conditioned media from either large scale COS7 transfections(pSVF8-80KG) or from transfected (amplified) CHO cell lines (pSVF8-80Kcell line lOC2B5; pSVF8-80A, cell line A1N; pSVF8-80S, cell line SlR)were prepared. The medium was DME H12 with 10% FBS. FVIII-LC waspurified for sequencing by a two-step procedure comprising ion exchangechromatography followed by affinity chromatography. Ion exchangechromatography was performed as follows: A column of S-FF Sepharose® (15×0.8 cm) was equilibrated with 0.02M MES, 0.05M NaCl, 0.01M CaCl₂, pH5.8, λ₂₀° C. =7.2 mS. Conditioned medium (500-1300 mL) was applied tothe column after adjustment of pH to 5.8 with a flow rate of 100 mL/h.The column was washed with 10 column volumes of 0.05M imidazole, 0.05MNaCl, 0.01M CaCl₂, pH 7.35, λ₂₀° C. =8.8 mS at a flow rate of 200 mL/h.FVIII-LC was eluted by addition of 0.1M CaCl₂ to the washing buffer,flow rate 50 mL/h. All operations were performed at 4° C.

Affinity chromatography was performed as follows: The murine monoclonalanti-FVIII-LC antibody 56-IgG was coupled to Sepharose® 4B by the CNBrmethod to a density of 2.5 mg/mL gel. The FVIII-LC containing eluate wasincubated with the immunosorbent overnight at room temperature, 1 mL ofgel per 1000 units FVIII-LC. The gel was then packed into a column andwashed with 20 column volumes of a low salt buffer (0.05M imidazole,0.15M NaCl, 0.01M CaCl₂, 10% glycerol, 0.02% NaN₃ pH 7.3), followed by20 column volumes of a high salt buffer (0.05M imidazole, 1.0M NaCl, 10%glycerol, pH 7.3). FVIII-LC was eluted from the immunosorbent using 1MCaCl2 in 0.05M imidazole, 0.15M NaCl, 10% glycerol after one hourincubation. The eluate was immediately desalted on a Sephadex® G-25column to a solution of 0.05M imidazole, 0.15M NaCl, 0.01M CaCl₂ 10%glycerol, 0.02% Tween® 80, 0.02% NaN₃, pH 7.3 and stored at -80° C.N-terminal sequence analysis was performed on an Applied Biosystem 477Asequencer.

The results of this analysis are shown in Table 7. The 80K glycoproteinencoded by pSVF8-8OKG has a tripeptide extension on its aminoterminus.Presumably this is the tPA pro tripeptide Gly-Ala-Arg, which cannot beprocessed by the Arg-Glu protease that recognizes the FVIII:C B domain.Further, the N-terminal sequences reveal that the signal peptide of tPAis actually 22 amino acid residues in length, with signal peptidasecleavage occurring on the carboxy side of Pro₂₂. Therefore, plasmidconstructions pSVF8-80S and pSVF8-80A, predicated upon signal peptidasecleavage after Ser₂₃ and Ala₂₈, respectively, lead to incorrect aminoterminal residues on the 80K light chains.

                                      TABLE 7                                     __________________________________________________________________________    N-terminal Sequences of 80 K Chains                                           with Modified tPA pre-pro Regions                                                                             Amount                                        Plasmid                                                                              N-terminal Sequence      (pmol)                                        __________________________________________________________________________    pSVF8-80                                                                             X--Ile--X--Arg--Thr--X--Leu--Gln--X--Asp--Gln--                                                        10                                            pSVF8-80KG                                                                           X--X--Arg--Glu--Ile--Thr--Arg--Thr--Thr--Leu--                                                         20                                            pSVF8-80S                                                                            Ser--Glu--Ile--Thr--Arg--Thr--                                                                         40                                            pSVF8-80A                                                                            X--Gln--Glu--Ile--       40                                            __________________________________________________________________________

Results shown in this example reveal the difficulty of predicting how asecreted polypeptide will be processed following transcription andtranslation. Modifications of the protein sequence have unexpectedconsequences for proteolytic processing and oligosaccharide addition,and can affect the overall efficiency of secretion.

Example 6

This example describes a method for expression of authentic FVIII:Clight chains using the signal peptide of human α₁ -antitrypsin.

A. Preparation of plasmids

1. pSVα1AT.Met A cDNA encoding the mature human α₁ -antitrypsinpolypeptide had been assembled using fragments of human liver cDNAclones and a synthetic oligonucleotide; the assembly was ligated as aBamHi-SalI fragment into pBR322 to make plasmid pAT(Met) (Rosenberg etal, Nature (1984) 312:77-80). A synthetic oligonucleotide linker-adapterand part of a cDNA clone encoding the signal peptide were used to attachthe signal peptide coding sequence, with an EcoRI restriction site onthe 5' end, to the BamHI site of pAT(Met). The resulting 1271 bpEcoRI-SalI fragment, encoding the translated sequences of human α₁-antitrypsin, was ligated into the EcoRI-SalI sites of pSV7d (describedin Example 1) to make pSVα1AT.Met.

2. pSVF8-80AT

Plasmid pSVα1AT.Met was opened at the BamHI site, which occurs at theboundary between the codons of the signal peptide and mature α₁-antitrypsin sequences. The cohesive end of this restriction site wasremoved with mung bean nuclease to leave the GAG (Glu) codon, and the α₁-antitrypsin sequence was deleted by digestion with SalI. The codingsequence of FVIII:C 80K was prepared for attachment by in vitromutagenesis of codons 1 and 2 of pSVF8-80 to form an EcoRV site (whichpreserves codon 2 as an Ile codon). This allowed the FVIII:C light chaincoding sequence (as an EcoRV-SalI sequence starting at codon 2) to befused in correct reading frame to codon 1 of α₁ -antitrypsin, andreplace the coding sequence of mature human α₁ -antitrypsin.

The coding sequence of pSVF8-80AT in the region of fusion is showntranslated below. Except for substitution of the a₁ -antitrypsin signalpeptide coding sequence for the tPA pre-pro coding sequence, thisplasmid is identical to pSVF8-80.

    __________________________________________________________________________    pSVF8-80AT (amino terminal region):                                           __________________________________________________________________________    -24         -20            -15            -10                                 Met                                                                              Pro                                                                              Ser                                                                              Ser                                                                              Val                                                                              Ser                                                                              Trp                                                                              Gly                                                                              Ile                                                                              Leu                                                                              Leu                                                                              Leu                                                                              Ala                                                                              Gly                                                                              Leu                                 ATG                                                                              CCC                                                                              TCG                                                                              AGC                                                                              GTC                                                                              TCG                                                                              TGG                                                                              GGC                                                                              ATC                                                                              CTC                                                                              CTG                                                                              CTG                                                                              GCA                                                                              GGC                                                                              CTG                                             -5             1           5                                      Cys                                                                              Cys                                                                              Leu                                                                              Val                                                                              Pro                                                                              Val                                                                              Ser                                                                              Leu                                                                              Ala                                                                              Glu                                                                              Ile                                                                              Thr                                                                              Arg                                                                              Thr                                                                              Thr                                 TGC                                                                              TGC                                                                              CTG                                                                              GTC                                                                              CCT                                                                              GTC                                                                              TCC                                                                              CTG                                                                              GCT                                                                              GAG                                                                              ATC                                                                              ACT                                                                              CGT                                                                              ACT                                                                              ACT                                          10             15             20                                     Leu                                                                              Gln                                                                              Ser                                                                              Asp                                                                              Gln                                                                              Glu                                                                              Glu                                                                              Ile                                                                              Asp                                                                              Tyr                                                                              Asp                                                                              Asp                                                                              Thr                                                                              Ile                                                                              Ser                                 CTT                                                                              CAG                                                                              TCT                                                                              GAT                                                                              CAA                                                                              GAG                                                                              GAA                                                                              ATT                                                                              GAC                                                                              TAT                                                                              GAT                                                                              GAT                                                                              ACC                                                                              ATA                                                                              TCA                                 __________________________________________________________________________

B. Expression and amino acid sequence analysis

1. Expression of DSVF8-80AT in COS7 cells

COS7 cells were transfected with pSVF8-80AT and a heavy chain expressionplasmid, usually pSVF8-92C. Conditioned media were assayed by LC-ELISA,HC-ELISA and COATEST. Transfected cells were also labeled withradioactive Met, so that the biosynthetically radiolabeled FVIII:C lightchains could be immunoprecipitated and visualized after polyacrylamidegel electrophoresis. Plasmid SVF8-80AT directs the synthesis of FVIII:Clight chains that appear as a doublet of 77-80K M_(r) . The amountproduced in COS7 cells is the same as for pSVF8-80. Co-expression withpSVF8-92C, or other FVIII:C heavy chain plasmid, leads to production ofactive FVIII:C complexes measured in the COATEST assay.

2. Purification and amino acid sequence analysis

Material for purification was prepared by transfection of COS7 cells inT-175 flasks, using increased cell density and decreased chloroquinediphosphate concentration. Conditioned media were collected 60 hoursafter transfection. Purification and amino acid sequence analysis wereperformed as described in Example 5. The results of aminoterminalsequence analysis (Table 8) indicate that the FVIII:C light chainencoded by PSVF8-8OAT has the same aminoterminal sequence as authentichuman plasma FVIII:C light chain.

                                      TABLE 8:                                    __________________________________________________________________________    N-terminal Sequence of 80 K Chains Secreted                                   Using α.sub.1 -antitrypsin Signal Peptide                                                                Amount                                       Plasmid                                                                              N-terminal Sequence       (pmol)                                       __________________________________________________________________________    pSVF8-80                                                                             X--Ile--X--Arg--Thr--X--Leu--Gln--X--Asp--Gln--                                                         10                                           pSVF8-80AT                                                                           Glu--Ile--Thr--Arg--Thr--X--Leu--Gln--Ser--Asp--Gln                                                     10                                           __________________________________________________________________________

3. In vitro assembly of 80AT FVIII:C light chains

The ability of 80 AT FVIII:C light chains to recombine with purifiedFVIII:C heavy chains in vitro was tested in an experiment shown in Table9. Purified FVIII:C light chains were incubated at concentrations of 3.7U/mL with purified recombinant (from full-length human FVIII:C) heavychains at 17 U/mL in buffer containing 50 mM Mn⁺⁺ and 150 μMp-mercapto-ethanol. As the control, purified recombinant heavy and lightchains were allowed to reassociate under the same conditions, and thequantity of active FVIII:C produced was assayed by COATEST. Theseresults suggest that the 80AT FVIII:C light chain can be combined invitro with purified recombinant heavy chain.

                  TABLE 9                                                         ______________________________________                                        Combination of Recombinant FVIII:C Light                                      Chains with Heavy Chains in vitro                                             Purified      Purified Percent Control                                        FVIII-LC      FVIII-HC Activity                                               ______________________________________                                        80AT          full-length                                                                            86                                                     80S           full-length                                                                            51                                                     80A           full-length                                                                            92                                                     80KG          full-length                                                                            233                                                    ______________________________________                                    

Example 7

This example describes plasmids for improved expression of the FactorVIII:C heavy chain. Modifications in DNA sequences responsible for theinitiation of transcription and in non-coding sequences are made inorder to increase the efficiency of transcription and the stability ofthe messenger RNA. The heavy chain glycoprotein is modified by acarboxy-terminal extension composed of segments of the B domain joinedby a short peptide. This is done to obtain a heavy chain that issecreted from cells more efficiently, is more stable in tissue culturemedium, and assembles more efficiently with the light chain.

A. Preparation of plasmids

1. pCMVF8-92/6x

In an effort to improve the level of transcription and stability of themessenger RNA for the Factor VIII:C 92K M_(r) heavy chain, the SV40early transcriptional initiation region was replaced by sequences fromthe human cytomegalovirus immediate early region (Boshart et al, Cell(1985) 4:521-530). In addition, 5' untranslated sequences contributed bythe SV40 early region to the messenger RNA were replaced with the 5'untranslated sequences of the HCMV lEl gene, including its first intron.This intron is included on the assumption that spliced transcripts leadto faster processing and more stable MRNA. The expression vector alsohas an SV40 origin of replication to permit transient expression in COS7cells, and a bacterial β-lactamase gene to permit DNA cloning byselection for ampicillin resistance.

The plasmid was constructed from a 700 bp SalI-PvuI fragment of pSV7d(described in Example 1) containing the SV40 polyadenylation region, a1400 bp PvuI-EcoRI (filled in with Klenow polymerase) fragment of pSVT2(Myers et al, Cell (1981) 25:373-84; Rio et al, Cell (1983) 32:1227-40)providing the SV40 origin of replication and the rest of the β-lactamasegene, a 1700 bp SspI-SalI fragment derived from a plasmid subclone ofthe human cytomegalovirus (Towne strain) in which the SalI site wasintroduced by in vitro mutagenesis near the translational start site forthe 1E1protein, and the 4300 bp SalI-SalI fragment of pSVF8-92C(described in Example 2) containing the cDNA encoding the Factor VIII:C92K M_(r) glycoprotein.

2. PSVF8-92tβ

This plasmid is a derivative of pSVF8-92C that encodes the 92K M_(r)recombinant heavy chain with a C-terminal extension composed ofN-terminal and C-terminal amino acid residues of the central (B) domainof the Factor VIII:C precursor linked by a peptide hinge peptidehomologous to that of human immunoglobulin a heavy chain. It is composedof a 4900 bp HindIII-SalI fragment from pSVF8-92C, into which wasinserted a 110 bp HindIII-SalI synthetic linker-adapter (shown below).##STR4## The linker-adapter encodes a carboxy-terminal extension of 34additional amino acid residues, and one potential site of N-linkedglycosylation. The C-terminal peptide should increase the molecularweight of the heavy chain to approximately 96K M_(r) , and to about 99KM_(r) if it is glycosylated.

B. Assay for FVIII:C heavy chain antigen and FVIII:C complex formation

The cofactor activity of the FVIII:C light chain-heavy chain complex wasestimated using a commercially available test kit from KabiVitrum(COATEST). Immunoreactive FVIII:C light chain was measured by ELISAusing HZ IgG coating antibody and peroxidase-conjugated antibodies fromNordisk Gentofte. The FVIII:C heavy chain immunoreactivity wasquantified using an ELISA developed at Nordisk Gentofte, which employshuman polyclonal antibody from an inhibitor patient (E-IgG).

C. Transient expression of nCMVF8-92/6x

The pCMVF8-92/6x plasmid was cotransfected with various FVIII:C lightchain plasmids (described in Example 5) into COS7 cells using theDEAE-dextran procedure. A sample of results from these transfections isshown in Table 10. The data suggest that addition of the CMV1E1promoter/enhancer, and the 5' untranslated sequences of the 1E1geneyields a 2.5 fold improvement (on average) in FVIII:C heavy chainexpression.

                  TABLE 10                                                        ______________________________________                                        Expresslon in COS7 Cells of                                                   PCMVF8-92/6x Versus pSVF8-92C                                                           FVIII: C Activity (mU/mL)                                           HC-Plasmid  LC-Plasmid   COA.sup.a                                                                             HC-RIA.sup.b                                 ______________________________________                                        PSVF8-92C   pSVF8-80     46      160                                          pSVF8-92C                                                                     80A         34            72                                                  PSVF8-92C                                                                     80R         61           310                                                  PSVF8-92C                                                                     80S         31            46                                                  pCMVF8-92/6x                                                                  80          87           290                                                  PCMVF8-92/6x                                                                  80A         131          140                                                  PCMVF8-92/6x                                                                  80R         178          330                                                  pCMVF8-92/6x                                                                  80S         114          690                                                  ______________________________________                                         .sup.a COATEST assay                                                          .sup.b Radioimmunoassay for heavy chain                                  

D. Transient expression in COS7 Cells of pSVF8-92tβ

Shown in Table 11 are the results of cotransfecting pSVF8-92tβ with aFactor VIII:C light chain expression plasmid (pSVF8-80AT, described inExample 6) into COS7 cells. The 92tβ heavy chain is secreted at higherlevels than the 92C heavy chain, which has a single amino acid (Ser)carboxy-terminal extension. The ratio of COATEST (COA) activity toELISA-reactive glycoprotein (a measure of complex formation) is greaterfor 92tβ chains than for 92C chains. In addition, the 92tβ heavy chainis secreted well in serum-free medium and appears to be stable, with aratio of activity to protein nearly the same as in 10% FBS. Theseresults show that this 34 amino acid carboxy-terminal extension improvessecretion and stabilizes the recombinant FVIII:C heavy chain.

                                      TABLE 11                                    __________________________________________________________________________    Expression of pSVF8-92tβ in COS7 Cells                                                          mU/mL FVIII                                            Exp.sup.†                                                                  Medium                                                                             Plasmid 1                                                                            Plasmid 2                                                                            COATEST                                                                              LC.sup.a                                                                         HC.sup.b                                     __________________________________________________________________________    1   10% FBS                                                                            pSVF8-92tβ                                                                      pSVF8-80AT                                                                           41     487                                                                              98                                                    pSVF8-92C                                                                            pSVF8-80AT                                                                           23     442                                                                              49                                           2   10% FBS                                                                            pSVF8-92tβ                                                                      pSVF8-80AT                                                                           80     484                                                                              162                                                   pSVF8-92C                                                                            pSVF8-80AT                                                                           30     639                                                                              90                                           3   HB CHO                                                                             pSVF8-92tβ                                                                      pSVF8-80AT                                                                           38     262                                                                              125                                          __________________________________________________________________________     .sup.† COS7 cell monolayers in duplicate were exposed to DNA in        DEAEDextran, washed, and treated with medium containing chloroquine           diphosphate for 8 hrs. Cells were washed to remove the drug, then covered     with 5 mL DME H21 containing 10% FBS. For expression in serumfree medium,     dishes were washed after 12-16 hrs. and overlaid with HB CHO ® from       Hana Biologicals. Conditioned media were assayed for FVIII:C activity as      described.                                                                    .sup.a by ELISA assay specific for light chain.                               .sup.b by ELISA assay specific for heavy chain.                          

Example 8

This example describes a method for expression of a FVIII:C heavy chainhaving Arg₇₄₀ as the C-terminus.

A. Preparation of plasmid pCMVF8-92R

The FVIII:C heavy chain encoded by the plasmid pCMVF8-92/6x has Ser₇₄₁as a C-terminal extension. In order to obtain a FVIII:C heavy chain withArg₇₄₀ as the C-terminus, a 1588 bp BamHI fragment of pCMVF8-92/6x ,encoding the 3' end of the coding sequence derived from pSVF8-92C waspurified. This fragment was cloned into ml3mpl8 and the Ser₇₄₁ residuewas changed to a translational stop codon by in vitro mutagenesis. ThepCMVF8-92R expression plasmid was assembled by cloning the mutagenizedBamHI fragment into the 5840 bp BamHI fragment of the original vector.By this procedure 680 bp of FVIII:C 3' untranslated sequences weredeleted.

B. Transient expression in COS7 cells of pCMVF8-92R

The pCMVF8-92R plasmid was co-transfected with the FVIII:C light chainplasmid pSVF8-80AT (described in Example 6) into COS7 cells using thecalcium phosphate technique (Graham and van der Eb, Virol (1973)52:456-67). The media were changed 18 and 42 hours post-transfection.Media samples for assays were collected 66 hours post-transfection. Theresults from these assays are shown in Table 12 below. The data showsthat FVIII:C activity was generated when pCMVF8-92R was co-transfectedwith a plasmid providing expression of FVIII LC.

                  TABLE 12                                                        ______________________________________                                        Coexpression of pCMVF8-92R and pSVF8-80AT.                                              COA          HC:Ag    LC:Ag                                         Transfection                                                                            (mU/mL)      (mU/mL)  (mU/mL)                                       ______________________________________                                        A         243          400       990                                          B         263          460      1190                                          ______________________________________                                    

Example 9

This example describes the preparation of a stable CHO cell line thatproduces the native FVIII:C M_(r) 92K-80K complex.

The DHFR⁻ CHO cell line DG44 (G. Urlaub et al, Som Cell Mol Genet (1986)12:555-66) was first transfected with the plasmid pCMVF8-80AT. In thisplasmid the CMV promoter (described in Example 7) regulates FVIII LCcDNA derived from pSVF8-80AT (described in Example 6), and downstreamcontains the Ad-MLP/dhfr cassette derived from pAd-DHFR (described inExample 4). The cells were transfected using the polybrene methoddescribed by W. Chaney et al, Som Cell Mol Genet (1986) 12:237-44. Byselecting for DHFR⁺ cells (in DMEM+10% DFSC) several FVIII-LC producerswere isolated; one of which was designated 11W.

In order to introduce FVIII-HC into 11W, the cell line was cotransfectedwith the plasmid pPR78 (this plasmid is analogous to pCMVF8-80AT, butcontains the HC cDNA derived from pCMVF8-92R described in Example 8instead of the LC cDNA) and pSV2-neo (P. J. Southern and P. Berg, J MolAppl Gen (1982) 1: 327-41). The transfection method used was themodified calcium phosphate procedure (G. Chen and H. Okayama, Mol CellBiol (1987) 7:2745-52). Transfectants were isolated in medium containing700 μg/mL Geneticin (G418 sulfate, Gibco). Cells from the primary poolwere subcloned by limiting dilution, and individual clones tested forexpression of active FVIII:C. In this way several FVIII:C producing celllines were isolated, one of which was designated 45-4/B-9.

45-4/B-9 was grown to confluency in a T-75 culture flask (DMEM+10%DFCS+700 μg/mL Geneticin) at 37° C., after which the cells weretransferred to 27° C. and acclimatized for 3 days before an expressionperiod of 24 hours. After the expression period, the FVIII:Cconcentration was measured to 12.8 U/mL using the chromogenic assay(Kabi).

Deposit Information:

The following materials were deposited with the American Type CultureCollection:

    ______________________________________                                        Plasmid      Deposit Date                                                                              Accession No.                                        ______________________________________                                        pSVF8-92     24 January 1986                                                                           40222                                                pSVF8-80     24 January 1986                                                                           40223                                                pSVF8-200    17 July 1985                                                                              40190                                                ______________________________________                                    

The above materials have been deposited with the American Type CultureCollection, Rockville, Md, under the accession numbers indicated. Thisdeposit will be maintained under the terms of the Budapest Treaty on theInternational Recognition of the Deposit of Microorganisms for purposesof Patent Procedure. The deposits will be maintained for a period of 30years following issuance of this patent, or for the enforceable life ofthe patent, whichever is greater. Upon issuance of the patent, thedeposits will be available to the public from the ATCC withoutrestriction.

These deposits are provided merely as convenience to those of skill inthe art, and are not an admission that a deposit is required under 35U.S.C. §112. The sequence of the polynucleotides contained within thedeposited materials, as well as the amino acid sequence of thepolypeptides encoded thereby, are incorporated herein by reference andare controlling in the event of any conflict with the writtendescription of sequences herein. A license may be required to make, use,or sell the deposited materials, and no such license is granted hereby.

Although the foregoing invention has been described in some detail byway of illustration and example for purposes of clarity ofunderstanding, it will be obvious that certain changes and modificationsmay be practiced within the scope of the appended claims.

What is claimed:
 1. A method for producing a recombinant protein complexhaving human Factor VIII:C activity, said complex comprising a firstpolypeptide homologous to the A domain of human Factor VIII:C, and asecond polypeptide homologous to the C domain of human Factor VIII:C,and further wherein the complex lacks all or a portion of the B domainof human Factor VIII:C, which method comprises:co-expressing in aeukaryotic host cell(a) a first polynucleotide that comprises apylynucleotide encoding the first polypeptide comprising a first aminoacid sequence homologous to the A domain of human Factor VII:C, and (b)a second polynucleotide that comprises a polynucleotide encoding thesecond polypeptide comprising a second amino acid sequence homologous tothe C domain of human Factor VIII:C, wherein neither the first nor thesecond polynucleotide encodes the complete B domain of human FactorVIII:C and further wherein the protein complex has coagulation activity.2. A method according to claim 1, wherein not more than about 5% of theamino acid residues of the first and second amino acid sequences differfrom the naturally occurring amino acid sequence of the Factor VIII:C Aand C domains, respectively.
 3. A method according to claim 1, whereinthe second amino acid sequence is the same as the amino acid sequence ofamino acid residues 1649-2322 of human Factor VIII:C.
 4. The method ofclaim 3, wherein the first amino acid sequence is the same as the aminoacid sequence of amino acid residues 1-740 of human Factor VIII:C. 5.The method of claim 3, wherein the first amino acid sequence is the sameas the amino acid sequence of amino acid residues 1-1102 of human FactorVIII:C.
 6. The method of claim 3, wherein the first amino acid sequenceis the same as the amino acid sequence of amino acid residues 1-1315 ofhuman Factor VIII:C.
 7. The method of claim 3, wherein the first aminoacid sequence is the same as the amino acid sequence of amino acidresidues 1-1405 of human Factor VIII:C.
 8. The method of claim 1,wherein said first polvnucleotide further comprises a polylinker.
 9. Themethod of claim 8 wherein the first polynucleotide and secondpolynucleotide are in separate expression plasmids.
 10. The method ofclaim 8, wherein said first polypeptide comprises the first 978 aminoacids of native Factor VIII:C and a C-terminal extension of 4 aminoacids encoded by the polylinker.
 11. The method of claim 1 wherein saidfirst polypeptide further comprises a C-terminal extension derived froma human Ig heavy chain hinge region.
 12. The method of claim 11 whereinsaid human Ig heavy chain hinge region is a human IgA1 heavy chain hingeregion.
 13. The method of claim 12 wherein the amino acid sequence ofthe C-terminal extension comprises Pro-Pro-Thr-Pro-Pro-Thr.
 14. Themethod of claim 1 wherein the first polynucleotide further comprises:(a)a 5' untranslated DNA sequence that increases the expression of thefirst polypeptide, wherein said 5'0 untranslated sequence is positioned5' to said polynucleotide encoding the first polypeptide, wherein said5' untranslated DNA is selected from the group consisting of humanFactor VIII:C 5' untranslated DNA, SV40 t antigen 5' untranslated DNA,and human cytomegalovirus 1E1protein 5'0 untranslated DNA; or (b) a 3'untranslated DNA sequence that enhances the expression of the firstpolypeptide, wherein said 3' untranslated sequence is positioned 3' tosaid polynucleotide encoding the first polypeptide, wherein said 3'untranslated DNA is selected from the group consisting of human FactorVIII:C 3' untranslated DNA, human tissue plasminogen activator 3'untranslated DNA, and SV40 t-antigen 3' untranslated DNA.
 15. The methodof claim 1 wherein the second polynucleotide further comprises:(a) a 5'untranslated DNA sequence that increases the expression of said secondpolypeptide, wherein said 5' untranslated sequence is positioned 5' tosaid polynucleotide encoding the second polypeptide, wherein said 5'untranslated DNA is selected from the group consisting of human FactorVIII;C 5'untranslated DNA, SV40 t antigen 5' untranslated DNA, and humancytomegalovirus 1E1protein 5' untranslated DNA; or (b) a 3' untranslatedDNA sequence that enhances the expression of the second polypeptide,wherein said 3' untranslated sequence is positioned 3' to saidpolynucleotide encoding the second polypeptide, wherein said 3'untranslated DNA is selected from the group consisting of human FactorVIII:C 3' untranslated DNA, human tissue plasminogen activator 3'untranslated DNA, and SV40 t-antigen 3' untranslated DNA.
 16. The methodof claim 1 wherein the eukaryotic host cell is a mamilian cell.
 17. Themethod of claim 1 wherein the first polynucleotide and secondpolynucleotide are in separate expression plasmids.
 18. The method ofclaim 1, wherein said first polynucleotide further comprises apolylinker and wherein said first polypeptide comprises the first 1315amino acid residues of native Factor VIII:C and a C-terminal extensionof 8 amino acid residues encoded by the polylinker.
 19. The method ofclaim 1, wherein said first polynucleotide further comprises apolylinker and wherein said first polypeptide comprises the first 1405amino acid residues of native Factor VIII:C and a C-terminal extensionof 11 amino acid residues encoded by the polylinker.
 20. The method ofclaim 1, wherein said first polynucleotide further comprises apolylinker and wherein said first polypeptide comprises the first 1102amino acid residues of native Factor VIII:C and a C-terminal extensionof 5 amino acid residues encoded by the polylinker.
 21. The method ofclaim 1 wherein said first polynucleotide further encodes a signalsequence capable of directing secretion.
 22. The method of claim 21wherein said signal sequence comprise the signal sequence of humanα1-anti-trypsin.
 23. The method of claim 1 wherein said secondpolynucleotide further encodes a signal sequence capable of directingsecretion.
 24. The method of claim 23 wherein said signal sequencecomprise the signal sequence of human α1-anti-trypsin.
 25. A DNAcomposition for transforming a eukaryotic host cell to obtainco-expression of a recombinant protein complex lacking all or a portionof the B domain of human Factor VIII:C having human Factor VIII:Cactivity, wherein said DNA composition comprises:a first expressioncassette comprising a first polynucleotide that comprises a firstnucleotide sequence encoding a first polypeptide comprising a firstamino acid sequence homologous to the A domain of human Factor VIII:C;and a second expression cassette comprising a second polynucleotide thatcomprises a second nucleotide sequence encoding a second polypeptidecomprising a second amino acid sequence homologous to the C domain ofhuman Factor, wherein neither the first nor the second polynucleotideencodes the complete B domain of human Factor VIII:C and further whereinthe DNA composition is effective in producing a protein complex havingcoagulation activity.
 26. The DNA composition of claim 25, wherein saidfirst polynucleotide further comprises a polylinker and wherein saidfirst polypeptide comprises the first 978 amino acid residues of nativeFactor VIII:C and a C-terminal extension of 4 amino acid residuesencoded by the polylinker.
 27. A host mammalian cell containing the DNAcomposition of claim
 26. 28. The DNA composition according to claim 25,wherein said first polynucleotide encodes a polypeptide comprising atleast about 90% of the amino acid sequence of human Factor VIII:C aminoacid residues 1-740 and said second cassette polynucleotide encodes apolypeptide comprising at least about 90% of the amino acid sequence ofhuman Factor VIII:C amino acid residues 1649-2332.
 29. A host mammaliancell containing the DNA composition of claim
 25. 30. The DNA compositionof claim 26, wherein said first polynucleotide further comprises apolylinker and wherein said first polypeptide comprises the first 1315amino acid residues of native Factor VIII:C and a C-terminal extensionof 8 amino acid residues encoded by the polylinker.
 31. The DNAcomposition of claim 25, wherein said first polynucleotide furthercomprises a polylinker and wherein said first polypeptide comprises thefirst 1405 amino acid residues of native Factor VIII:C and a C-terminalextension of 11 amino acid residues encoded by the polylinker.
 32. TheDNA composition of claim 25, wherein said first polynucleotide furthercomprises a polylinker and wherein said first polypeptide comprises thefirst 1102 amino acid residues of native Factor VIII:C and a C-terminalextension of 5 amino acid residues encoded by the polylinker.
 33. TheDNA composition of claim 25 wherein said first polynucleotide furtherencodes a signal sequence capable of directing secretion.
 34. The DNAcomposition of claim 25 wherein said second polynucleotide furtherencodes a signal sequence capable of directing secretion.